Von Willebrand Disease Type Research Articles

Semiautomatic VWF Multimer Densitometric Analysis: Validation of the Clinical Accuracy and Clinical Implications in Von Willebrand Disease

Semiautomatic VWF Multimer Densitometric Analysis: Validation of the Clinical Accuracy and Clinical Implications in Von Willebrand Disease

Traumatic Hyphema in a Type 1 Von Willebrand Patient: Clinical Case and Management

Von Willebrand disease (vWD) is the most common inherited congenital bleeding disorder. Type 1 is a deficiency, but not absence, of von Willebrand factor (vWF) and is the most common type. Bleeding episodes in patients with type 1 vWD vary and are related to the relative amount of von Willebrand factor produced. We report a case of a 15-year-old male with mild von Willebrand disease type 1 (vWF: 35%) who presented with a right eye grade lll hyphema and elevated intraocular pressure (IOP) following a traumatic injury. Despite medical management with Diamox, ophthalmic steroids, vWF replacement with Humate-P, the patient had a repeat bleeding event and worsening of his elevated intraocular pressure. Due to concern for corneal blood staining, the patient required surgical intervention with an anterior chamber washout. Von Willebrand factor replacement was given on presentation and prior to the procedure with goal von Willebrand factor (vWF) activity and Factor Vlll (FVlll) levels of 80-100 IU/dL. Factor replacement for a traumatic hyphema is necessary to reduce bleeding, but could lead to increased risk of thrombosis formation and increase risk of corneal staining. Balance between bleeding and thrombosis can lead to treatment challenges in patients with bleeding disorders including vWD. Disclosures Smink: Forma Therapeutics:Membership on an entity's Board of Directors or advisory committees;Highmark Insurance:Membership on an entity's Board of Directors or advisory committees.

Shear stress induced activation of von Willebrand factor may predispose to gastrointestinal bleeding in syndrome of Heyde

Abstract Introduction Von Willebrand factor (vWF) changes conformation from globular to fibrillar in the range of shear rates above 5000 s-1. High shear rates, observed in severe aortic stenosis, create conditions for activation of vWF, which opens up access for platelets and coagulation factors to the previously hidden domains of the molecule. At the same time, vWF undergoes increased degradation by metalloproteinase ADAMTS13. Proteolytic cleavage of vWF leads to deficiency of hemostatically active high molecular weight multimers (HMWM) of vWF, while its mass concentration remains unaltered. This results in the development of acquired von Willebrand disease type 2A (vWD) and concomitant gastrointestinal bleeding. The combination of acquired vWD 2A and gastrointestinal bleeding, developed due to severe aortic stenosis, is called Heyde's syndrome. The correlation of shear stress activation of vWF and gastrointestinal bleeding in patients with Heyde's syndrome remains poorly studied. The aim of the study was to measure vWF-mediated platelet adhesion to fibrinogen-coated surfaces under shear rates higher than 5000 s-1 in whole blood samples of healthy volunteers and patients with Heyde's syndrome. Methods A microfluidic system simulating blood flow in vessels was used to assess platelet adhesion. Platelet adhesion was measured by an increase in the intensity of laser radiation scattered from a fibrinogen-coated surface during a 15 minutes circulation of whole blood samples through a flow chamber under shear rates higher than 5000 s-1. Platelets in whole blood samples were activated with 5 μM ADP prior to measurement. The study included 5 patients with Heyde's syndrome 55–80 years old. The control group included 6 healthy volunteers 25–55 years old. vWF-mediated platelet adhesion was detected by blocking platelet-vWF binding with anti-GPIb monoclonal antibody (mAb). Fibrinogen-mediated platelet adhesion was detected by blocking platelet GPIIb/IIIa receptors with mAb. Result The inhibition of GPIb vWF-receptor reduced platelet adhesion by 7.6±3.5% (p<0.05) in patients with Heyde's syndrome, and by 16.5±3.3% (p<0.05) in healthy volunteers. The inhibition of GPIIb/IIIa fibrinogen receptor reduced platelet adhesion by 96±7% (p<0.05) in patients with Heyde's syndrome, and by 80.2±6.6% (p<0.05) in healthy volunteers. Conclusion Significantly reduced contribution of vWF to platelet adhesion under shear rates higher than 5000 s-1 may indicate a decrease in hemostatically active HMWM of vWF. Shear stress activation of vWF in the range of high shear rates and its subsequent inactivation by ADAMTS13 may lead to functional vWF deficiency and the development of gastrointestinal bleeding in Heyde's syndrome. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Russian Science Foundation

Comparison of von Willebrand factor platelet‐binding activity assays: ELISA overreads type 2B with loss of HMW multimers

BackgroundA number of new assays with different measuring principles are available to measure von Willebrand factor (VWF) glycoprotein Ib (GPIb)‐binding activity, but little is known about how these assays might behave differently for subtypes of von Willebrand disease (VWD). ObjectivesThe Comparison of Assays to Measure VWF Activity (COMPASS‐VWF) study was designed to compare all available VWF GPIb‐binding activity assays for VWF. We specifically searched for particular assay behavior differences. Patients/MethodsTo sort out random differences from systematic assay behavior deviations, all assays were performed in different laboratories on the same samples in a blinded fashion. Samples from 53 normal controls and 42 well‐characterized VWD patients were reanalyzed in this study to dissect assay‐specific discrepancies. ResultsNo assay behavior differences were found for 53 normal controls. For VWD patients, we found the following systematic assay behavior patterns: (a) All ELISA assays for VWF:GPIbR as well as VWF:GPIbM are insensitive to detect the low VWF activity of VWD type 2B patients with loss of high molecular weight multimers; (b) VWF:Ab assay reports higher activity for the p.V1665E mutation than all other assays; and (c) all ristocetin‐based assays (including VWF:RCo using fixed platelets) but the AcuStar assay report discrepantly low VWF activity for the p.P1467S polymorphism. No systematic assay‐specific difference was observed for either the particle agglutination VWF:GPIbM assay or the AcuStar assay using magnetic beads. ConclusionsDifferent assay principles may lead to discrepant results for certain VWD types or mutations. Therefore, a more extensive study for a large number of patients is needed to better characterize the incidence and relevance of such assay‐specific differences.

Von Willebrand disease type 2N in a neonate: A case report

The occurrence of von Willebrand disease (VWD) is relatively uncommon. Type 1 is more prevalent than the other types. The disease prevalence is about only 1%, in which type 1 includes 60–80% of the cases, type 2 includes 20–30% of cases, and type 3 accounts for <5% of the cases. It mainly arises due to homozygous or composite heterozygous mutations in the von Willebrand factor gene (12p13.3) leading to synthesis of a truncated protein or allele silencing. We report a case of newborn 34-week gestation who presented with abdominal distension with melena and seizures. The child’s plasma was studied and it revealed VWD.

Population Pharmacokinetic Modeling of von Willebrand Factor Activity in von Willebrand Disease Patients after Desmopressin Administration.

Most von Willebrand disease (VWD) patients can be treated with desmopressin during bleeding or surgery. Large interpatient variability is observed in von Willebrand factor (VWF) activity levels after desmopressin administration. The aim of this study was to develop a pharmacokinetic (PK) model to describe, quantify, and explain this variability. Patients with either VWD or low VWF, receiving an intravenous desmopressin test dose of 0.3 µg kg-1, were included. A PK model was derived on the basis of the individual time profiles of VWF activity. Since no VWF was administered, the VWF dose was arbitrarily set to unity. Interpatient variability in bioavailability (F), volume of distribution (V), and clearance (Cl) was estimated. The PK model was developed using 951 VWF activity level measurements from 207 patients diagnosed with a VWD type. Median age was 28 years (range: 5-76), median predose VWF activity was 0.37 IU/mL (range: 0.06-1.13), and median VWF activity response at peak level was 0.64 IU/mL (range: 0.04-4.04). The observed PK profiles were best described using a one-compartment model with allometric scaling. While F increased with age, Cl was dependent on VWD type and sex. Inclusion resulted in a drop in interpatient variability in F and Cl of 81.7 to 60.5% and 92.8 to 76.5%, respectively. A PK model was developed, describing VWF activity versus time profile after desmopressin administration in patients with VWD or low VWF. Interpatient variability in response was quantified and partially explained. This model is a starting point toward more accurate prediction of desmopressin dosing effects in VWD.

Management of von Willebrand disease with a factor VIII‐poor von Willebrand factor concentrate: Results from a prospective observational post‐marketing study

BackgroundA triple‐secured plasma‐derived von Willebrand factor (pdVWF) almost devoid of factor VIII (FVIII):WILFACTIN®, was approved in France in 2003, and then in other countries for the treatment of patients with von Willebrand disease (VWD). ObjectiveTo investigate long‐term safety and efficacy of the product in real‐life over the first 5 post‐approval years. Patients/MethodsThis prospective, observational, national post‐marketing study (PMS) enrolled patients of all ages and VWD types. Patients were observed for up to 3 years and treated for one or more occasions. Efficacy was assessed for each major event. Breakthrough bleeding rate 3 days post‐infusion and annualized bleeding rate (ABR) were also evaluated for long‐term prophylaxis. ResultsOverall, 155 of 174 patients enrolled from 31 centers were eligible for efficacy assessment. Most patients (76.8%) were severely affected (VWF:RCo ≤ 15 IU/dL). They were treated for 743 bleeds and 140 surgeries including childbirth. Efficacy outcomes were excellent/good for 98.2% of 56 major surgeries and 94.0% of 67 major bleeds. Approximately 75% of 49 major mucosal bleeds were effectively managed without FVIII co‐administration. In 32 patients receiving prophylaxis, breakthrough bleeding occurred in 1.5% of infusions and median ABR was 1.0 for 20 patients treated ≥ 12 months. Excellent tolerability was confirmed with no safety concerns. No thrombotic events were observed. ConclusionsResults from this PMS increase the clinical experience of a FVIII‐poor pdVWF in patients of all ages and VWD types including those with thrombotic risk factors and emphasize that giving FVIII is not always mandatory to effectively treat patients with severe VWD.

Detection of acquired von Willebrand syndrome after ventricular assist device by total thrombus-formation analysis system.

AimsBleeding is a serious complication in patients with continuous‐flow left ventricular assist device (CF‐LVAD). Acquired von Willebrand syndrome (AVWS; type 2A) develops because of high shear stress inside the pumps and is a cause of bleeding complication. Although von Willebrand factor (vWF) multimer analysis is useful for diagnosing AVWS, it is only performed in specialized research institutes. A novel microchip flow chamber system, the total thrombus‐formation analysis system (T‐TAS), is a point‐of‐care system to evaluate the thrombus‐formation process and useful for monitoring platelet thrombus‐formation capacity in patients receiving antiplatelet therapy and the diagnosis and evaluation of the clinical severity of von Willebrand disease type 1. However, little is known about the association between AVWS and platelet thrombus‐formation capacity evaluated by T‐TAS in patients with CF‐LVAD. We aimed to evaluate the utility of T‐TAS for easy detection of AVWS in patients with CF‐LVAD.Methods and resultsWe simultaneously evaluated the vWF large multimers and T‐TAS parameters in four consecutive patients with axial‐type CF‐LVAD and eight control patients treated with aspirin and warfarin. vWF large multimer index was defined as the proportion of large multimers in total vWF derived from a normal control plasma. T‐TAS analyses different thrombus‐formation processes using two microchips with different thrombogenic surfaces. PL24‐AUC10 levels in the platelet (PL) chip are highly sensitive for platelet functions, while AR10‐AUC30 levels in the atheroma (AR) chip allow the assessment of the overall haemostatic ability. vWF large multimer index and T‐TAS parameters were decreased in all patients with CF‐LVAD. The mean PL24‐AUC10 level (5.4 ± 2.9 vs. 219 ± 67; P < 0.01), AR10‐AUC30 level (338 ± 460 vs. 1604 ± 160; P < 0.01) and vWF large multimer index (49 ± 11% vs. 112 ± 27%; P < 0.01) were significantly lower in the patients with CF‐LVAD than in control patients. One patient showed changes in T‐TAS levels before and after implantation of CF‐LVAD. PL24‐AUC10 and AR10‐AUC30 levels decreased from 438.1 to 5.0 and from 1667.9 to 1134.3, respectively.ConclusionsIn patients with CF‐LVAD, the platelet thrombus‐formation capacity was extremely impaired because of AVWS, and T‐TAS parameters could detect the presence of AVWS. T‐TAS can be used for easy detection of AVWS as a point‐of‐care testing. Further studies with a large sample size are needed to validate our results in several LVAD models and evaluate the prognostic value of bleeding complications and thromboembolism in patients with LVAD.

Are Iranian patients with von Willebrand disease type 2N properly differentiated from hemophilia A and do they receive appropriate treatment?

: The defect function of the von Willebrand factor (VWF) in carrying factor VIII (FVIII) leads to von Willebrand disease type 2N (VWD 2N) which could be easily misdiagnosed as hemophilia A. Differentiating of VWD 2N from hemophilia A is crucial for patient treatment and genetic counseling. As a retrospective study, we aimed to evaluate the current diagnostic work-up of Iranian patients with mild/moderate deficiency of FVIII levels and the possibility of misdiagnosis of VWD 2N as hemophilia A. All patients who referred to the reference coagulation laboratory at the Iranian Blood Transfusion Organization in a 10-months period for bleeding diathesis work-up with the request of FVIII activity level were included. Clinical and laboratory phenotypes including International Society on Thrombosis and Hemostasis - Bleeding Assessment Tool, FVIII activity, VWF antigen, VWF ristocetin cofactor, and FVIII binding capacity of VWF were assessed on suspected cases for VWD 2N. In total, the results of 896 patients for investigation of VWD 2N were evaluated and five new patients were identified within unrelated families with abnormal VWF:FVIIIB levels. Four were heterozygous for VWD 2N and one homozygous whom all were misdiagnosed as hemophilia A and underwent inappropriate treatments. The median bleeding score of the VWD 2N population was nine (4-13). In Iran, probably a significant number of VWD 2N patients are misdiagnosed as hemophilia A due to insufficient test panel for subtyping of von Willebrand disease. This study also emphasized the need for inclusion of the VWF:FVIIIB in suspected hemophilia A to achieve an optimal treatment strategy.

Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms

AbstractCopy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.

Von Willebrand disease: Diagnosis and treatment, treatment of women, and genomic approach to diagnosis.

von Willebrand disease (VWD) is the most common inherited bleeding disorder. VWD is caused by deficiencies in von Willebrand factor (VWF), a critical adhesive haemostatic protein. This review provides an overview of VWD diagnosis and treatment, special considerations in treating women with VWD, and current genomic approaches to VWD. For diagnosis and treatment in VWD, an accurate diagnosis is critical to providing effective treatments, determining appropriate laboratory monitoring and for counselling the patient and family. Diagnosis of VWD begins with the clinical assessment for the bleeding phenotype, which is usually characterized by mucocutaneous and provoked bleeding. The diagnosis of VWD is then made by laboratory investigation. Multiple assays are used to assess VWF levels and functions. The mainstays of VWD treatment are tailored by VWD type and symptoms, and can include antifibrinolytic treatment, desmopressin and VWF replacement treatment. Women with VWD are also at risk for excessive uterine bleeding, such as with menses and childbirth. In addition to standard VWD treatments, heavy menstrual bleeding can be treated with hormones. Interdisciplinary management of childbirth and prophylaxis in the postpartum period are needed to reduce the risk of postpartum haemorrhage. Genomic approaches to VWD can inform VWD diagnosis, treatment, test assay selection, reproductive planning and family counselling. Most VWD patients have an identifiable VWF gene DNA variant. Next-generation sequencing is rapidly being adopted to provide more comprehensive VWF sequence information for patients with known or suspected VWD.

Comparative assessment of von Willebrand factor multimers vs activity for von Willebrand disease using modern contemporary methodologies.

Diagnosis of von Willebrand disease (VWD) is challenging due to heterogeneity of VWD and test limitations. Many von Willebrand factor (VWF) assays are utilized, including antigen (Ag), activity and multimer analysis. Activity assays include ristocetin cofactor using platelets (VWF:RCo) or other particles incorporating recombinant glycoprotein I ('VWF:GPIbR'), or other GPI binding assays using gain-of-function mutations ('VWF:GPIbM'), or collagen binding (VWF:CB). To comparatively evaluate modern contemporary VWF activity assays vs VWF multimer analysis using modern contemporary methods. Several VWF activity assays (VWF:RCo, VWF:GPIbR, VWF:GPIbM, VWF:CB) assessed (typically as a ratio against VWF:Ag) against a new semi-automated procedure for different types of VWD (1, 3, 2A, 2B, 2M), plus control material (n=580). The evaluation also focussed on relative loss of high and very high molecular weight multimers (HMWM and VHMWM) by densitometric scanning. All evaluated VWF activity/Ag ratios showed high correlation to the presence/absence of HMWM and VHMWM, although VWF:CB/Ag and VWF:GPIbR/Ag ratios using an automated chemiluminescence method yielded highest correlation coefficients (r=.909 and .874, respectively, for HMWM). Use of the investigative procedure (VHMWM) identified fewer false positives for 'loss' in type 1 VWD. This comparative investigation identified that new automated chemiluminescence VWF activity assays best identified relative loss or presence of HMWM and VHMWM according to activity to Ag ratios and an alternative investigative method for identifying VHMWM in multimer testing for a new commercial multimer method may lead to fewer false identifications of HMW loss in type 1 VWD.

Evaluation of a fully automated von Willebrand factor assay panel for the diagnosis of von Willebrand disease.

von Willebrand disease (VWD) diagnosis starts with first level tests: factor VIII coagulant activity, VWF antigen (VWF:Ag) and platelet-dependent VWF activity (VWF:RCo, VWF:Ab, VWF:GPIbR or VWF:GPIbM). The VWF collagen binding (VWF:CB) assay measures the binding capacity of von Willebrand factor (VWF) to collagen. To assess, in previously diagnosed VWD patients, the performance of a fully automated chemiluminescent test panel including VWF:Ag, VWF:GPIbR and VWF:CB assays. The patients, historically evaluated using in-house VWF:Ag and VWF:CB assays and an automated latex enhanced immunoassay VWF:GPIbR method, were re-evaluated using the VWF test panel HemosIL AcuStar. The VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag obtained by means of AcuStar showed an overall good concordance with the corresponding data obtained at the time of the historical diagnosis. When discrepancies occurred, these were generally due to the lower VWF:CB/VWF:Ag obtained with AcuStar as compared with that obtained with the historical methods and this affected particularly the diagnosis of VWD type 2M. Together, the AcuStar VWF:GPIbR/VWF:Ag and VWF:CB/VWF:Ag were able to distinguish type 1 from types 2A, 2B and 2M, whereas no distinction was possible between type 2A and 2B. The AcuStar panel offers a good performance in the differential diagnosis between VWD type 1 and 2A/2B patients. A high rate of coincidence with historical diagnosis was obtained for VWD types 3, 2A/2B and 1. Even though in some cases more tests (eg, RIPA/multimeric analysis) are needed to complete an accurate VWD classification, the AcuStar panel is considered a sensitive, rapid and reliable tool to diagnose VWD patients.

Dental invasive procedures in von Willebrand disease outpatients treated with high purity FVIII/VWF complex concentrate (Fanhdi®): experience of a single center

PurposeTo retrospectively assess the effectiveness and safety of customized hemostatic protocols using a plasma-derived, von Willebrand Factor (VWF)-containing Factor VIII concentrate (pdVWF/FVIII) in von Willebrand disease (VWD) patients undergoing dental invasive procedures. MethodsProtocol for each patient was drawn up by the Blood Unit based on the VWD type, disease severity, and type of treatment. pdFVIII/VWF infusions and doses were registered at 30–60 min before intervention (t0) and at 12-24-36-48-72 h after intervention (t12-t72) and up to day 7. Any peri- or postoperative bleeding, complication or adverse event was registered. ResultsForty-five dental procedures were performed on 20 VWD patients (six type-1, two type-2a, six type-2b, six type-3). Most pdFVIII/VWF infusions at t0 were 60 IU/kg (n = 7) and 50 IU/kg (n = 9). Subsequent infusions were mostly 30–50 IU/kg. No bleeding complications or adverse events were reported. ConclusionThis study supports the safety and efficacy of pdFVIII/VWF to prevent peri- and postoperative bleeding after invasive oral procedures.

Clinical significance of diagnostic algorithm in detection of mild hemostasis disorders in women with menorrhagia

Background/Aim. Coagulation disorders could be a cause of menorrhagia in women of reproductive age. The aim of the study was to estimate frequency of coagulation disorders and design an appropriate algorithm for detection of coagulation disorders. Methods. We investigated coagulation in 115 women (36.1 ? 9.6 years) with anamnestic data of menorrhagia, verified using semiquantitative method ? Pictorial Bleeding Assessment Chart (PBAC) with score ? 100. Results. Menorrhagia was objectively verified in sixty-four women (55.7%) and in comparison with those with normal menstruation they had higher PBAC score of menstrual cycle [median (Md) = 150.0 vs. Md = 50.0; p &lt; 0.001] but not its duration (7.2 ? 2.1 days vs. 7.3 ? 1.9 days; p &gt; 0.05). Coagulation defects was found in 12 (10.4%) women ? decreased F IX: Ac in 4 (3.5%), decreased F VII: Ac in 1 (0.9%), decreased F X: Ac in 1 (0.9%), decreased F XI: Ac in 1 woman (0.9%), while 5 (4.3%) women matched criteria for mild von Willebrand disease (VWD) type 1. Women with coagulation disorders had prolonged prothrombin time (PT) [Md = 13.1 s, range: 12.2?14.8 s vs. Md = 12.5 s, range 10.6?18.3 s; p = 0.032]. Anemia was diagnosed in 61 (53.0%) women. The strongest predictor of the hemostasis disorder was menorrhagia (Quotient of probability 0.018), then anemia presence (12.43), P? (2.35), menstrual cycle duration (1.16) and the PBAC score (0.98). Conclusion. The results of the study indicate the need to form a diagnostic algorithm for hemostasis disorders, primarily VWD. Sophisticated analysis of hemostasis is required, especially if predictive factors of statistical models are detected: objectively verified menorrhagia, anemia, prolonged menstrual cycle, PBAC score &gt; 100 and extended PT.

Genetic Background of von Willebrand Disease: History, Current State, and Future Perspectives.

Sequencing of the gene encoding for von Willebrand factor (VWF) has brought new insight into the physiology of VWF as well as its pathophysiology in the context of von Willebrand disease (VWD). Molecular testing in VWD patients has shown high variability in the overall genetic background of this condition. Almost 600 mutations and many disease-causing mechanisms have been described in the 35 years since the VWF gene was identified. Genetic testing in VWD patients is now available in many centers as a part of the VWD diagnostic algorithm. Molecular mechanisms leading to types 2 and 3 VWD are well characterized; thus, information from genetic analysis in these VWD types may be beneficial for their correct classification. However, the molecular basis of type 1 VWD is still not fully elucidated and most likely represents a multifactorial disorder reflecting a combined impact of environmental and genetic factors within and outside of VWF. Regarding sequencing methods, the previous gold-standard Sanger sequencing is gradually being replaced with next-generation sequencing methods that are more cost- and time-effective. Instead of gene-by-gene approaches, gene panels of genes for coagulation factors and related proteins have recently become a center of attention in patients with inherited bleeding disorders, especially because a high proportion of VWD patients, mainly those with low VWF plasma levels (type 1), appear to be free of mutations in VWF. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are accessible in a very limited number of laboratories. Results from these studies have presented several genes other than VWF or ABO possibly affecting VWF levels, and such findings will need further validation studies.

Expression of angiogenic factors in the uteroplacental unit is altered at time of placentation in a porcine model of von Willebrand disease type 1

Von Willebrand disease (VWD) affects blood coagulation and correlates with angiodysplasia. Data on VWD-affected women point to slightly increased miscarriage rates. We aimed to investigate the impact of VWD on angiogenesis in the uteroplacental unit of pregnant pigs of a model of VWD type 1 (T1). Uteri, placentae, and embryos were harvested at time of placentation (day 29 to 31) from four sows (two wildtype (WT) and two heterozygous for a von Willebrand factor (VWF) mutation diagnosed with T1). T1 sows were bred to a T1 boar creating embryos of three different genotypes: WT, T1 or homozygous for the VWF mutation corresponding with VWD type 3 (T3). Uteroplacental tissues were examined histologically. Embryos were genotyped. Gene expression of angiogenic factors possibly related to VWF was determined by quantitative real-time PCR. Corresponding protein expression was analyzed by immunohistochemistry. Genotyping revealed 35.3% WT, 52.9% T1 and 5.9% T3 embryos (5.9% not classified confidently). No histological alterations were found. Gene expression of VEGF was significantly increased in T1 placentae while expression of ANG1, ANG2, TIE2, and ITGB3 was significantly reduced, confirmed on protein level for different cell types. TIE2/TIE1 ratios were significantly lower in T1 placentae. Distribution of embryo genotypes indicates selection favoring the WT. Significant expression differences of angiogenic factors in placentae suggest influence of VWF on these factors during placentation, although angiodysplasia was not observed. The alterations concerning VEGF/VEGFR-2 signaling, integrin expression and the ANG/TIE system may influence angiogenesis and vascular adaptation during placentation and thus the overall outcome of pregnancy.

Prevalence, Clinical Characteristics and Outcome of Von Willebrand Disease in Oman

Introduction: Von Willebrand Disease (VWD) is the second most common inherited bleeding disorder. There is paucity of the literature describing the prevalence and clinical characteristics of VWD in this part of the world. The aim of the current study is to detect the prevalence, describe the spectrum of the different types of VWD, their mode of presentation, bleeding phenotype and outcome in Oman. Methods: A retrospective cross-sectional study was carried out in the 2 available referral tertiary care facilities in Oman namely; Sultan Qaboos University and the Royal Hospitals. The study included all children and adults diagnosed with VWD in Oman until June 2019. The patients were subtyped as per the International Society of Thrombosis and Haemostasis (ISTH) criteria. Data was collected from the electronic hospital systems in both hospitals. Out of 700 entries of VWD in both hospitals, only 140 were true cases and 560 were tested negative but wrongly labelled. Patients or their next of ken were called and interviewed to obtain the necessary information that was not documented in the electronic system. Results: A total of 140 patients are confirmed to have VWD giving a prevalence of 1:20000. Fifty eight patients are males (41.5%), 82 patients are females (58.5%). Sixty six patients have type I (47%), 38 patients have type II (27%) and 36 patients have type III (26%). The majority of patients 90 (64%) were diagnosed before the age of 20 years and 62 of them (68%) had positive family history of the disease. The most common presentation was recurrent unexplained bruising. As expected, patients with type III tend to have a significant bleeding phenotype with a bleeding score more than 5 in adults and 3 for paediatric patients. All of them were admitted to hospital at some point electively (for surgery) or for bleeding control, however, they were not put on prophylaxis. None of the patients had serious or intra-cranial bleeding. Conclusion: Von Willebrand Disease is not uncommon in Oman with an overall prevalence of 1:20000, however, it is much less than what was originally reported in previous studies in developing countries and the WFH website. The majority of patients are type 1 and have a positive family history of the disease. The disease is more common in females. All patients with type III have abnormal bleeding score and required VW factor replacement at one point. None of the patients had a serious bleed and they are not on prophylaxis. Keywords: Von Willebrand disease, Prevalence, Oman. Disclosures Al-Khabori: AstraZeneca: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; SOBI: Honoraria; NovoNardisk: Membership on an entity's Board of Directors or advisory committees; Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Altered Angiogenic Gene Expression Profiles in Porcine Von Willebrand Disease

Objectives: Von Willebrand Factor (VWF) is a main factor of coagulation, but also known for its influence on angiogenesis by modifying the expression of several genes. The mechanisms were assumed to base on shared genetic pathways and also on an altered storage capability of certain proteins within the VWF-dependent Weibel-Palade Bodies (WPBs). As women affected by Von Willebrand Disease (VWD), a disease caused by a deficiency of VWF, may develop miscarriage and other complications during pregnancy which highly relies on proper angiogenesis, we analyzed the effect of VWD on the expression of genes potentially influenced by a deficiency of VWF within uterus tissue. Methods: Uterus tissue samples were taken from each two non-pregnant VWD pigs suffering from VWD type 1 (T1, heterozygous) and type 3 (T3, homozygous), resp., during late estrus. Two healthy pigs served as controls (WT, wild type). The genes F8 (coagulation factor VIII), FGA (fibrinogen alpha chain), FGB (fibrinogen beta chain), FGG (fibrinogen gamma chain), PLG (plasminogen), CXCL8 (C-X-C motif chemokine ligand 8), CALR (calreticulin), CTGF (connective tissue growth factor), and IGFBP7 (insulin like growth factor binding protein 7) were chosen for analysis and specific primers were designed. The expression of these genes was analyzed by qRT-PCR and relatively quantified against the endothelial specific housekeeping genes PROCR and CD31 using the ΔΔCT method and the Wilcoxon-Mann-Whitney statistic test. The differences in gene expression were compared among the genotypes. Results: F8, FGA, FGG, PLG, CXCL8, CTGF, and IGFBP7 showed no significantly altered expression profiles. However, genes showing differential expression with significant (p ≤ 0.05) or suggestive (p ≤ 0.1) p-values among the three genotypes in uterus tissue were CALR and FGB. CALR expression was reduced to 36.7% and 33.3% within uterus tissue of T1 and T3 pigs, respectively, compared to WT (p ≤ 0.1). When combining the VWD pigs of both types and comparing them to WT, their expression was reduced to 35.0% reaching significance (p ≤ 0.05). The expression of FGB was increased to about five-fold in T1 pigs compared to the WT (p ≤ 0.1), while it was not significantly changed in T3 individuals. Discussion and Conclusions: CALR protein is stored in the VWF-dependent WPBs and shows physical interactions with VWF. As a reduction of VWF goes along with a reduction of WPBs, adequate CALR release might be diminished. Additionally, CALR is known to inhibit endothelial cell proliferation and to strongly affect angiogenesis. FGB shows strong physical interactions with CALR. It is part of the coagulation system and a homozygous dysfunction of this gene causes afibrinogenemia and consequently bleeding disorders. The observed higher expression of FGB in T1 uterus tissue compared to the WT might therefore reflect a higher production of fibrinogen. However, this needs to be confirmed in further studies. The observed reduced expression of CALR in uterus tissue of VWD-affected pigs might therefore contribute to the antiangiogenic alterations observed in VWD patients and contribute to the pregnancy related complications in VWD. Disclosures No relevant conflicts of interest to declare.

Clinical Study to Investigate the Efficacy and Safety of Wilate during Prophylaxis in Previously Treated Patients with Von Willebrand Disease (VWD)

Background: Inherited von Willebrand disease (VWD) is the most common inherited hemorrhagic disorder, with an estimated prevalence of 1 in every 100 individuals. Type 1 and type 3 (the most severe form) are characterized by a quantitative deficiency of von Willebrand factor (VWF) and type 2 arises from a qualitative deficiency of VWF. Treatment of VWD depends on the type and severity of the disease. Severe bleeding is reported in patients with all subtypes, leading to progressive joint disease as well as diminished quality of life (QoL). VWF/factor VIII (FVIII) concentrates have become the mainstay of VWD treatment for these patients with severe disease or for those patients in whom other treatments (e.g., desmopressin) are ineffective or contraindicated but this is broadly applicable only for on demand treatment. Aims: The primary objective of this study is to determine the efficacy of VWF/FVIIII concentrate in the prophylactic treatment of previously treated patients with type 3, type 2 (except 2N), or severe type 1 VWD. Secondary objectives of this study will be to collect data to 1) Assess the VWF:Ac and VWF:Ag incremental IVR of VWF/FVIIII concentrate over time and, 2) Assess the safety and tolerability of VWF/FVIIII concentrate in this indication. The study will also examine, the efficacy of VWF/FVIIII concentratein the treatment of breakthrough bleeding episodes (BEs), and in surgical prophylaxis, as well as the QoL during prophylaxis with VWF/FVIIII concentrate. Methods: The study is planned to enroll 28 patients aged ≥6 years and with VWD type 1, 2A, 2B, 2M, or 3. Eligible patients must be receiving on-demand treatment with a VWF-containing product, with at least 1, and an average of ≥2, documented spontaneous BEs per month in the preceding 6 months requiring treatment with a VWF-containing product. This will be assessed as part of a run-in observational study to collect the bleeding profile prior to the start of prophylaxis. From the beginning of the study, patients will receive prophylactic treatment with VWF/FVIIII concentrate for 12 months and record all BEs in a patient diary. Based on these data, the frequency of BEs and the annualized bleeding rate (ABR) under prophylactic treatment will be calculated. Treatment efficacy of BEs will be assessed by the patient (together with the investigator in case of on-site treatment) using a 4-point scale (excellent, good, moderate, none) In patients that undergo surgeries, efficacy of VWF/FVIIII concentratewill be assessed at the end of surgery by the surgeon and at the end of the postoperative period by the haematologist. In both cases, predefined assessment criteria will be used. In addition, an overall assessment of efficacy will be made at the end of the postoperative period by the investigator. Results: Data will be monitored on an ongoing basis and the study is expected to end Q2 2021. Conclusions: Prophylactic treatment in other congenital bleeding disorders is widely accepted as the standard of care to prevent bleeding and preserve QoL in patients but to date, this form of treatment in VWD is not well characterized. This study will provide data on the efficacy of prophylactic treatment in reducing the rate of bleeding and on the impact of prophylaxis on the QoL in VWD patients. Disclosures Sidonio: Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Research Funding; Uniqure: Membership on an entity's Board of Directors or advisory committees. Schwartz:Octapharma: Employment.