Phase II Biologic Effects Trial of Recombinant Interleukin-11 (rhIL-11, Neumega) in Moderate or Mild Hemophilia A or Von Willebrand Disease Unable to Use DDAVP,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3308-3308
Author(s):  
Margaret V. Ragni ◽  
Enrico M. Novelli ◽  
Anila Murshed ◽  
Elizabeth P. Merricks ◽  
Mark T. Kloos ◽  
...  
Keyword(s):  
Phase Ii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Postoperative Bleeding ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Fluid Restriction ◽  
Biologic Effects ◽  
Von Willebrand

Abstract Abstract 3308 Background: DDAVP is the treatment of choice for individuals with type 1 von Willebrand disease (VWD), although 20% are unresponsive, and of the 80% who do respond, the VWF increase is transient, as endothelial stores are depleted after 3 days. Further, administration requires a 30- minute intravenous infusion in a medical facility. Plasma-derived concentrates may be used in these settings, but are more costly and have potential risk of transmissible infection. We recently demonstrated that recombinant human IL-11 (rhIL-11, Neumega®), a gp-130 signaling cytokine with hematopoietic and anti-inflammatory activity, increases VWF activity up to 2-fold when given daily by subcutaneous injection, with levels persisting each day it is given, and reduces menstrual and postoperative bleeding. The effects of rhIL-11 in individuals with VWD unresponsive or allergic to DDAVP, or hemophilia A, however, have not been evaluated. Methods: We conducted a phase II trial to evaluate the safety and biologic effects of rhIL-11 in VWD patients unresponsive or allergic to DDAVP (VWD-Un) or mild hemophilia A (HemA). rhIL-11 was given subcutaneously at 25 μg/kg daily for 4 days in the non-bleeding state, followed on day 4, 30 minutes after rhIL-11, by one dose of DDAVP intravenously, 0.3 μg/kg, if not contraindicated (pt. 2). Fluid restriction was recommended. Fluid status was assessed by height, weight, and exam. Pre- and post-dosing laboratory assays included the VWD profile, VWF multimers by SDS gel electrophoresis, and platelet VWF mRNA by qPCR. Results: The results of the first six subjects, including three with VWD (one type IIB and two type 1 VWD), VWF:RCo 0.10–0.20 U/ml, and three with mild hemophilia A, F.VIII 0.08–0.12 U/ml, are presented. All subjects were healthy, with no hypertension or cardiac disease, and all had normal physical exams and normal EKGs. By day 4, among VWD-Un subjects, there was a 1.2-fold increase in VWF:RCo (15±3% vs. 12±0%); a 1.6-fold increase in VWF:Ag (22±8% vs.14±6%); and a 1.3-fold increase in VIII:C (34±36% vs. 27±10%), as compared with pre-rhIL-11 levels (Figure). Following DDAVP (except pt. 2), there was an additional 2.0-fold, 1.7-fold, and 2.6-fold increase in VWF:RCo, VWF:Ag, and VIII:C, respectively. Among HemA subjects, by day 4, there was a 1.8-fold increase in VWF:RCo (160±25% vs. 88±12%); a 1.8-fold increase in VWF:Ag (182±28% vs.99±18%), p<0.01; and a 1.5-fold increase in VIII:C (21±8% vs. 14±5%), as compared with pre-rhIL-11 levels. Following DDAVP, there was an additional 1.5-fold (p<0.01), 1.7-fold, and 2.8-fold (p<0.05) increase in VWF:RCo, VWF:Ag, and VIII:C, respectively. The drug was well tolerated well with less than grade 1 mild conjunctival erythema, local erythema and tenderness at the injection site; in one subject transient hyponatremia, Na 129 meq/L, occurred after excess oral fluid intake for diabetic hyperglycemia, which resolved with fluid restriction. Discussion: These data suggest that rhIL-11 increases VWF and VIII levels modestly in VWD patients unresponsive/allergic to DDAVP, and in mild hemophilia A, suggesting the potential use in treatment of clinical bleeding in these disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phase II prospective open-label trial of recombinant interleukin-11 in desmopressin-unresponsive von Willebrand disease and mild or moderate haemophilia A

2013 ◽  
Vol 109 (02) ◽  
pp. 248-254 ◽  
Author(s):  
Enrico Novelli ◽  
Anila Murshed ◽  
Elizabeth Merricks ◽  
Mark Kloos ◽  
Timothy Nichols ◽  
...  
Keyword(s):  
Phase Ii ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Fluid Restriction ◽  
Haemophilia A ◽  
Open Label ◽  
Open Label Trial ◽  
Grade 3 ◽  
Von Willebrand ◽  
Interleukin 11

SummaryDesmopressin (DDAVP) is the treatment of choice in those with mild von Willebrand disease (VWD), yet 20% are unresponsive to DDAVP, and among the 80% who respond, the response is transient, as endothelial stores are depleted after three days. We, therefore, conducted a single-center Phase II clinical trial to determine safety and biologic efficacy of recombinant interleukin-11 (rhIL-11, Neumega®) in patients with VWD unresponsive or allergic to DDAVP, or mild or moderate haemophilia A (HA). Increases in VWF:RCo were observed by 48 hours after rhIL-11, with a 1.54-fold increase by Day 4, 1.30-fold in VWD and 1.73-fold in HA. Similarly, by 48 hours, increases in VIII:C were observed, with a 1.65-fold increase by Day 4, 1.86-fold in VWD and 1.48-fold in HA. Platelet VWFmRNA expression by qPCR increased 0.81-fold but did not correlate with plasma VWF:Ag responses. rhIL-11 was well tolerated, with grade 1 or less fluid retention, flushing, conjunctival erythema, except for transient grade 3 hyponatraemia in one subject after excess fluid intake for diabetic hyperglycaemia, which resolved with fluid restriction. In summary, rhIL-11 increases VWF levels in two of four DDAVP-unresponsive or allergic VWD and F.VIII levels in four of five mild or moderate haemophilia A subjects, suggesting its potential use in treatment of these disorders.

Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 541-541
Author(s):  
Giancarlo Castaman ◽  
Sofia Helene Giacomelli ◽  
Paula M. Jacobi ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Negative Impact ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Wild Type ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 541 Background. Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study. We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules. Methods. Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results. The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression. In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions. Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures: No relevant conflicts of interest to declare.

Intracellular Retention, Enhanced Clearance, and Defective FVIII Binding Are Common Features of Von Willebrand Factor D'-D3 Domain Mutations in Patients with Von Willebrand Disease Type 1 From the European Mcmdm-1VWD Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 99-99 ◽  
Author(s):  
Reinhard Schneppenheim ◽  
Ulrich Budde ◽  
Javier Batlle ◽  
Giancarlo Castaman ◽  
Jeroen C. J. Eikenboom ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Recombinant Expression ◽  
Von Willebrand Disease ◽  
Phenotypic Characterization ◽  
Clinical Markers ◽  
In Trans ◽  
Von Willebrand ◽  
In Cis

Abstract Abstract 99 Background: Von Willebrand disease (VWD) type 1 is characterized by a partial reduction of structurally and functionally normal VWF with normal VWF multimers. As part of a large European study (Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) patients previously diagnosed with VWD type 1 were studied systematically to assess the phenotypic and genotypic spectrum. Objective: To confirm the pathogenicity of VWF gene mutations and to elucidate the molecular mechanisms of VWD type 1. Patients and methods: VWD type 1 patients were recruited by twelve expert centers in nine European countries. VWF genotyping was performed in all index cases (IC). The eight mutations studied here are located in the VWF D'-D3 domain and corresponded to 57 patients from 19 families. They were reproduced by recombinant expression with subsequent phenotypic characterization, two of them in cis and one in trans with a second mutation. Results and Discussion: Intracellular VWF:Ag of all mutants was normal or near normal suggesting normal expression levels. However, seven mutations (p.M771I, p.I1094T, p.C1130R, p.C1130G, p.C1130F, p.W1144G and p.Y1146C) caused intracellular retention and impaired VWF secretion. In addition, we observed a major loss of high molecular weight multimers as in type 2A and a novel finding of a severe VWF:FVIII binding defect in most of the homozygously expressed mutants. Additional mutations either in cis or in trans had no modifying effect. The recombinant VWD type 1 Vicenza mutation p.R1205H with or without the allelic variant p.M740I seen in three Italian IC was secreted normally and had normal function leaving enhanced clearance of mutant VWF as the only pathomechanism. In conclusion, the majority of mutations in the D3 domain impair VWF multimerization, cause intracellular retention and correlate with defective FVIII binding. An elevated ratio of VWF propeptide to VWF:Ag suggests enhanced VWF clearance as an important pathomechanism of most mutations and particularly of p.R1205H. Disclosures: No relevant conflicts of interest to declare.

Experience with Epidural Anesthesia in Pregnant Women with Von Willebrand Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1026-1026
Author(s):  
Jay Varughese ◽  
Alice J. Cohen
Keyword(s):  
Pregnant Women ◽  
Von Willebrand Factor ◽  
Epidural Anesthesia ◽  
Postpartum Hemorrhage ◽  
Postoperative Bleeding ◽  
Von Willebrand Disease ◽  
Bleeding Complications ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand Disease (vWD) is an autosomal dominant inherited bleeding disorder that is characterized by epistaxis, mucosal and postoperative bleeding, menorrhagia and postpartum hemorrhage. In particular, there is a paucity of safety data for, and thus a reluctance to use, epidural anesthesia (EA) for delivery. We thus conducted a review of all women followed with vWD in a referral hemophilia clinic who had ≥ 1 pregnancy. Thirty-three subjects were screened; 31/33 (94%) had type 1 and 2/33 (6%) had type 2A vWD. There were 59 term pregnancies (range 1–3 per patient), and 5 fetal losses (in 4 patients). Of the term pregnancies, 16/59 (27%) were delivered by Caesarian Section (C-Section), complicated by postpartum hemorrhage in 3 (19%); 43/59 (73%) were delivered by normal spontaneous vaginal delivery (NSVD), complicated by hemorrhage in 21 (49%) (p=0.05). EA was administered during 14 (13 with type 1 vWD) of 59 (24%) of the deliveries, all without DDAVP, plama-derived factor VIII-von Willebrand factor containing concentrates or blood products, and in no patient were bleeding complications encountered at the site of EA nor were there any neurologic complications. Conclusion: Postpartum hemorrhage was a common complication in patients with vWD, more after NSVD than C-Section. In a selected subset, EA was safely administered without bleeding complications, possibly due to pregnancy induced increase in factor VIII:C and von Willebrand factor activity counteracting the tendency to bleed. Larger series and prospective studies should be performed to confirm the safety of EA and the relationship to coagulation factor levels in pregnant women with vWD.

Bleeding Score as a Preoperative Predictor of Postoperative Bleeding in Type 1 Von Willebrand Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1019-1019 ◽  
Author(s):  
Laura R. Goldberg ◽  
Margaret V. Ragni
Keyword(s):  
Family History ◽  
Postoperative Bleeding ◽  
Von Willebrand Disease ◽  
Chi Square ◽  
Exact Test ◽  
Bleeding Score ◽  
Chi Square Analysis ◽  
Bleeding History ◽  
Von Willebrand

Abstract Type 1 Von Willebrand Disease (VWD) is the most common congenital bleeding disorder, affecting 1% of the population, and caused by a quantitative deficiency of Von Willebrand Factor (VWF). In addition to mucosal bleeding, VWD patients often suffer postoperative bleeding, leading to significant morbidity. Thus, a preoperative diagnosis could potentially reduce postoperative bleeding. Because symptoms correlate poorly with VWD assays, subject to extragenic effects and lab variability, diagnosis is difficult. The bleeding score (BS) is a simple quantitative tool recently developed to rate bleeding symptom severity, with 99% specificity for VWD. To determine the potential utility of BS in predicting postoperative bleeding in VWD, we evaluated preoperative BS by retrospective review of type 1 VWD patients who suffered postoperative bleeding prior to diagnosis. Preoperative clinical bleeding symptoms and VWD assays, including VWF:RCo, VWF:Ag, and FVIII:C, were obtained. The severity of clinical bleeding symptoms present prior to surgery was rated by the 4-point BS scale: 0 = no/trivial; 1 = present; 2 = intervention required; 3 = replacement therapy. Statistical analysis was by chi square analysis and Fisher’s exact test for categorical data, and by student t test for continuous data. Of 260 registered type 1 VWD patients, 71 (27.3 %) experienced surgical bleeding prior to a diagnosis of VWD. Of these 56 (78.9%) were female, 48 (67.6%) were adults (≥ 18 yr), and 61 (85.9%) had a family bleeding history. The surgeries included general, gynecologic, genitourinary, and otolaryngologic procedures. The median preop BS, 3 in females and 4 in adults, was significantly higher than in males and children, each median 1, p&lt;0.01, respectively. A BS ≥ 3 would have identified only 59.1% patients before surgery, but as many as 90.1%, if combined with one abnormal VWD test; 94.4%, with family bleeding history; or 97.2% with both family history and one abnormal VWD test. The proportion of children identified by BS was significantly lower than in adults, 26.1% vs 75.0 % with BS &gt; 3, p = 0.001. Yet this significantly improved by combining BS with family history, 91.3% vs 95.8%, not different from adults, p = 0.591. We conclude that obtaining a preoperative BS and family bleeding history may reduce postoperative bleeding and promote timely diagnosis among individuals with type 1 VWD patients, particularly children. Preoperative Bleeding Score in Type 1 VWD Patients with Postoperative Bleeding Male Female Age &lt; 18 Age ≥ 18 All N = 15 N = 56 N = 23 N = 48 N = 71 τp = .001, as compared with under 18 yr; σp = .007, as compared with males; ζ p &gt; 0.5 as compared with age under 18 or males, respectively. BS≥1 10/15 (66.7%) 54/56 (96.4%) 18/23 (78.3%) 46/48 (95.8%) 64/71 (90.1%) BS≥3 4/15 (26.7%) 38/56 (67.8%)σ 6/23 (26.1%) 36/48 (75.0%)τ 42/71 (59.1%) BS≥5 2/15 (13.3%) 17/56 (30.3%) 2/23 (8.7%) 17/48 (35.4%) 19/71 (26.7%) Abnl VWF:RCo 8/15 (53.3%) 19/56 (33.9%) 6/23 (26.1%) 19/48 (39.6%) 27/71 (38.0%) Abnl VWD Test 11/15 (73.3%) 41/56 (73.2%) 16/23 (69.6%) 36/48 (75.0%) 52/71 (73.2%) Fam Bld History 15/15 (100%) 47/56 (83.9%) 21/23 (91.3%) 40/48 (83.3%) 61/71 (85.9%) BS≥3 ± Abnl VWD Test 13/15 (86.7%) 51/56 (91.1%) 18/23 (78.3%) 46/48 (95.8%) 64/71 (90.1%) BS≥3 ± Fam Hx 15/15 (100.0%) 52/56 (92.8%)ζ 21/23 (91.3%) 46/48 (95.8%)ζ 67/71 (94.4%) BS≥3 ± Fam Hx ± Abnl VWD Test 15/15 (100.0%) 54/56 (96.4%) 22/23 (95.6%) 47/48 (97.9%) 69/71 (97.2%)

Cost Effectiveness Strategy to Predict Postoperative Bleeding in Von Willebrand Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1295-1295
Author(s):  
Daniel Delitto ◽  
Margaret V. Ragni ◽  
Kenneth J Smith
Keyword(s):  
Cost Effectiveness ◽  
Incremental Cost ◽  
Postoperative Bleeding ◽  
Bleeding Risk ◽  
Von Willebrand Disease ◽  
Test Series ◽  
Surgical Bleeding ◽  
Bleeding History ◽  
Von Willebrand

Abstract Von Willebrand disease (VWD) is the most common congenital bleeding disorder, affecting up to 1% of the population. In type 1 VWD, which accounts for 65% of the disease, bleeding may be mild and up to 50% may have a normal screening APTT. Thus, in many the diagnosis may be missed and excess bleeding may occur postoperatively, which could potentially be prevented if reliable preoperative predictors existed. In order to assess possible predictors of postoperative bleeding and determine a cost-effective strategy to predict postoperative bleeding in patients with type 1 VWD, we constructed a decision tree model to test various screening strategies, including bleeding severity score (BSS), VWD assays (VWF:RCo; VWF:Ag, VIII:C), platelet function (PFA-100), and family bleeding history (Fam Hx), alone and in combination, in various sequence. For the model, sensitivity and specificity of coagulation tests, BSS, and family bleeding history were determined on 129 subjects with type 1 VWD, including 74 experiencing postoperative bleeding prior to diagnosis and 55 experiencing no postoperative bleeding prior to diagnosis. BSS was determined for each subject at the time of diagnosis (but prior to surgical bleeding), based on abstracted outpatient medical records. Testing, transfusion, and hospitalization costs were based on local costs, and DDAVP cost was based on average wholesale price. Outcomes were testing strategy cost and strategy effectiveness, as measured by postoperative bleeding risk. It was assumed that all patents testing positive, based on a given strategy, would receive preoperative DDAVP and be 100% protected from bleeding; and that those who developed postoperative bleeding would require three days of hospitalization and receive a transfusion. In the base case analysis, with VWD prevalence of 1%, BSS 3 3 and the Test Series (any of VWF:RCo VWF:Ag, F.VIII) dominates over all other strategies, based on having the highest sensitivity. When prevalence is varied 0–1%, the strategy of BSS 3 3 and the Test Series seems a reasonable choice if prevalence is 3 1%. At 1% prevalence, the incremental cost-effectiveness ratio (ICER) is about $103,000 which is at the edge of what may be considered economically reasonable. However, the ICER, for this strategy falls fairly rapidly, and is &lt; $100,000 when prevalence is 3 1.03%. We also varied the proportion requiring hospitalization or transfusion to 5% and 1%, respectively, but these did not significantly affect the results. In conclusion, in individuals with BSS 3 3, proceeding with the Test Series (VWF:RCo, VWF:Ag, F.VIII) is a clinically and economically reasonable strategy for those in whom DDAVP should be given to prevent surgical bleeding, when the bleeding risk is 3 1%. Strategy Cost Incremental Cost Effectiveness Incremental Effectiveness ICER BSS ≥ 3 & Test Series $1,296.10 0.055 Fam Hx $1,703.90 $407.70 0.087 −0.032 (Dominated) Fam Hx & Test Series $2,187.50 $891.40 0.111 −0.056 (Dominated) Fam Hx or BSS ≥ 3 & Test Series $2.435.40 $1,139.30 0.127 −0.072 (Dominated) Test Series $2,508.60 $1,212.50 0.132 −0.077 (Dominated) BSS ≥ 3 $2.974.30 $1,678.10 0.171 −0.116 (Dominated) BSS ≥ 5 $5,966.10 $4.669.90 0.364 −0.309 (Dominated) No Testing or Rx $9,238.30 $7.942.20 0.574 −0.519 (Dominated)

Molecular Analysis of Type 1 VWD Mutations: Effects of Mutant:Wild-Type Transfection Ratio and Protein Degradation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1210-1210
Author(s):  
Tara C White-Adams ◽  
Paula M Jacobi ◽  
Sandra L Haberichter ◽  
Jorge A Di Paola
Keyword(s):  
Conflicts Of Interest ◽  
Phenotypic Variability ◽  
Statistical Significance ◽  
Von Willebrand Disease ◽  
Incomplete Penetrance ◽  
Shear Stresses ◽  
Dependent Manner ◽  
Von Willebrand

Abstract Abstract 1210 Background: Von Willebrand disease (VWD), the most frequently diagnosed bleeding disorder, is characterized by variable expressivity and incomplete penetrance. Bleeding severity in type 1 VWD does not always correlate with plasma VWF levels, except in cases of severe deficiency. It is possible that the phenotypic variability observed in type 1 VWD is related to the final ratio of mutant vs. wild-type (WT) subunits in the mature VWF multimeric structure. The aim of this study was to determine the role of mutant:WT transfection ratio on von Willebrand factor (VWF) expression, secretion and degradation in VWD type 1 mutations. Methods: Type 1 VWD mutations with reported normal multimer distribution were chosen from the D'-D3 region of VWF. Mutations of cysteine residues were eliminated to avoid interference with inter- and intra-chain disulfide linkages. Mutations were generated by performing site-directed mutagenesis on full-length human VWF cDNA within the pcDNA3.1(-)A vector, which appends VWF with a Myc-His tag (denoted mH). The following mutations were generated: M771I, R782Q, R924W, I1094T and T1156M. Mutant VWF was co-transfected with WT VWF contained within the pCIneo vector (mutant mH:WT pCIneo ratios investigated were 1:3, 2:2, 3:1, 4:0). Recombinant (r)VWF expression was measured using ELISA and concentrations were determined by comparison to a standard curve generated with pooled normal plasma. Multimer composition was analyzed using SDS-agarose gel electrophoresis followed by Western blot. Statistical significance was determined using one-way ANOVA with post-hoc Tukey test. Results: Homozygous expression of R924W or I1094T had no effect on rVWF expression or secretion compared to WT, while M771I, R782Q and T1156M significantly increased intracellular protein retention. Co-expression of M771I or R782Q at varying ratios with WT was able to partially correct rVWF secretion, although intracellular retention remained significantly higher than WT at all ratios (n=3, * p<0.05, Figure 1). Co-expression with WT cDNA was also able to correct T1156M retention in a dose-dependent manner (n=3, Figure 1), as described previously [Lethagen, Thromb and Haemost, 2002]. Multimer analysis of co-transfection supernatants exhibited normal and full distribution of multimers, as expected for type 1 VWD mutations. Others have shown previously that heterodimers of WT and C1149R VWF, a type 1 VWD mutation, are degraded by the proteasome [Bodo et al, Blood, 2001], presumably via recognition of a folding defect within the mutant subunit. In order to determine the role of proteasomal degradation in the decreased secretion levels of our mutants, we performed experiments in the presence of the proteasome inhibitor MG-132. Treatment of co-transfected cells (mutant:WT 2:2) with 1 mM MG-132 for 16 hours prior to harvesting did not significantly affect secretion or overall expression of rVWF, suggesting that this pathway is not involved in the regulation of the expression of our mutants. Discussion: Our data demonstrate that M771I, T1156M and R782Q induce a significant increase in intracellular retention compared to WT protein, which could contribute to a quantitative deficiency in type 1 VWD, while R924W and I1094T do not appear to interfere with VWF production or secretion. Variable levels of intracellular retention have been observed in a previous study of VWF mutations identified in type 1 VWD patients [Eikenboom, et al, J Thromb Haemost, 2009]. While one interpretation of these results is that R924W or I1094T may not be causative mutations in type 1 VWD, other mechanisms including protein clearance and function remain to be explored. Although type 1 VWD mutations variably affect expression and secretion levels in vitro, studying platelet rolling on these mutants at a range of physiological shear stresses will provide valuable information regarding whether the degree of incorporation of mutant subunits into VWF multimers can affect supramolecular structure, and ultimately, hemostatic function. Disclosures: No relevant conflicts of interest to declare.

Desmopressin responsiveness at a capped dose of 15 μg in type 1 von Willebrand disease and mild hemophilia A

2014 ◽  
Vol 25 (8) ◽  
pp. 820-823 ◽  
Author(s):  
Dou-Anne Siew ◽  
Joy Mangel ◽  
Lori Laudenbach ◽  
Sheila Schembri ◽  
Leonard Minuk
Keyword(s):  
Hemophilia A ◽  
Von Willebrand Disease ◽  
Von Willebrand

scholarly journals Targeted DNA Sequencing for Hemophilia A, B, and von Willebrand Disease: Analytical Precision in Clinical Laboratories and Improved Disease Characterization

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5042-5042
Author(s):  
Patricia Severino ◽  
Liliane Santana Oliveira ◽  
Natalia Torres ◽  
Joao Carlos Guerra ◽  
Nelson Hamerschlak ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Sanger Sequencing ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Targeted Sequencing ◽  
Probe Design ◽  
Clinical Laboratories ◽  
Von Willebrand

Abstract Hemophilia A, B, and von Willebrand disease correspond to more than 90% of all inherited bleeding disorders associated with coagulation factor deficiencies. Symptoms between these deficiencies may vary greatly and yet are often phenotypically similar. Bleeding episodes can range from mild to severe, at times with life threatening hemorrhages. Currently, biochemical assays are performed to assess the function of each coagulation factor, but diagnosis remains cumbersome and prone to multiple sources of variability between laboratories. Genetic evaluation allows for the examination of multiple coagulation factor genes simultaneously and may quickly identify possible causes to the disease. Additionally, genetic testing should be more reproducible and readily comparable between clinical laboratories. In this work we evaluate the potential use of targeted sequencing of three coagulation factors genes – F8, F9 and VWF – for the concurrent diagnosis and characterization of hemophilia A, B, and von Willebrand disease samples. For targeted DNA sequencing we selected specific DNA probes using genomic coordinates spanning the complete intronic and exonic regions of the three genes, as well as flanking gene sequences. Eleven hemophilia A samples and four hemophilia B samples, clinically characterized and submitted to Sanger sequencing for F8 and F9 genes coding regions, respectively, were included in this study. Our results indicate that even though DNA quality may be ideal for traditional DNA sequencing, enrichment techniques require more intact fragments, as reflected by variations in sequencing coverage between samples: quadruplicate results per sample showed 100X coverage varying from 80% of sequenced regions to less then 20%. Point substitutions found in F9 genes by Sanger sequencing were confirmed by targeted sequencing, but results for F8 gene were less satisfactory, in agreement with probe design limitations at this point. Of interest for hemophilia A patients, four samples possessed, in addition to the alterations in F8, point mutations in VWF. Probe design and sequencing parameters did not allow for the identification of F8 intron 1 and intron 22 inversions, frequent alterations in hemophilia A, but optimization procedures are currently underway. We conclude that targeted sequencing approach may be a viable and more complete solution for the diagnosis and management of hemophilia A, B and von Willebrand disease. Disclosures No relevant conflicts of interest to declare.

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