Type 2M Research Articles

Comparison of a new chemiluminescent immunoassay for von Willebrand factor activity with the ristocetin cofactor-induced platelet agglutination method

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50IUdL(-1) ): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.

189 DEFECTIVE VON WILLEBRAND FACTOR AND ANGIOPOIETIN-2 RELEASE FROM VON WILLEBRAND DISEASE PATIENTS’ BLOOD OUTGROWTH ENDOTHELIAL CELLS

Von Willebrand factor (VWF) mediates platelet adhesion to damaged endothelial cells (EC) and stabilises blood coagulation factor VIII. In EC, VWF is stored within and directs the formation of Weibel-Palade bodies (WPB), which contain many proteins including the angiogenic regulator Angiopoietin (Ang)-2. Von Willebrand disease (VWD), the most common inherited bleeding disorder is caused by decrease (type 1 and 3) or dysfunction (type 2) of VWF. VWD can be associated with angiodyplasia, describing the presence of vascular lesions which can cause intractable bleeding. We demonstrated a role for VWF in angiogenesis and hypothesized that angiodysplasia might be linked to dysregulated angiogenesis through several pathways. VWD is a heterogenous disorder and a wide range of mutations in the VWF gene have been characterised; however their cellular consequences are still poorly understood. We have used a recently developed approach to study the molecular and cellular basis of VWD and angiogenesis. Mononuclear cells were isolated from peripheral blood of four type 1 VWD, four type 2 VWD patients and nine healthy controls and cultured to obtain blood outgrowth EC (BOEC). We confirmed the endothelial lineage of BOEC by staining for various EC markers. Confocal microscopy confirmed VWF and Ang-2 co-staining within BOEC WPB. We measured VWF mRNA and protein levels, both before and after stimulation, and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis (Pearson R=0.9976, p=0.0024**, r²=0.9952). However BOEC from this group of patients also showed defects in processing, storage and/or secretion of VWF. Two patients with type 1 VWD showed increased levels of pro-peptide containing VWF one of which showed expansion of the endoplasmic reticulum consistent with retention of defective VWF.Three type 1 patients showed no Ang-2 release post-cellular stimulation, consistent with the inability of these BOEC to correctly package WPB. One patient showed significant release of Ang-2 (P&lt;0.05); interestingly this patient showed the highest levels of VWF within the type 1 group with more WPB and VWF strings released upon stimulation. Levels of VWF mRNA and protein were normal in BOEC from three type 2 VWD patients, supporting the dysfunctional VWF model. However, one type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. All type 2 patients’ BOEC show a significant release of Ang-2 upon stimulation, confirming correct packaging of WPB. Together our data suggest that the angiogenesis defect in type 1 VWD patients is due to dysregulation of Ang-2 storage and release, but that other mechanisms may be more relevant in type 2 VWD. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into the cellular mechanisms of the disease.

Viola tricolor (Violaceae) is a karyologically unstable species

Viola tricolor is a pseudometallophyte covering heavy-metal-polluted and non-polluted areas. The species is a member of the evolutionarily young sect. Melanium of Viola. In this study, we sought to determine whether the karyotype of V. tricolor is stable with respect to chromosome structure or is altered depending on environmental conditions (heavy-metal-polluted vs. non-polluted areas). We established the karyotypes of plant material originating from a Zakopane meadow (non-metallicolous population) and from the Bukowno mine waste heap (metallicolous population), showing evident interpopulation differentiation in chromosome type (2M+20 m+2sm+2st vs. 18 m+8sm), in the number, size, and distribution of rDNA loci (25S and 5S), and also in chromosome mutations, mainly fission of chromosomes into acentric fragments and translocation of the fragments. Variable numbers of both 25S and 5S rDNA loci were distributed at different positions of the chromosomes and not on specific pairs of chromosomes. The results clearly indicate that the karyotype of V. tricolor results from the unstable genetic structure of the species. This character, typical for relatively young evolutionary groups, proves its membership to the Melanium section considered to be young within the genus Viola.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells

AbstractVon Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

Variability in platelet‐ and collagen‐binding defects in type 2M von Willebrand disease

Type 2M von Willebrand disease (VWD) includes qualitative defects in von Willebrand factor (VWF) function, with normal multimer distribution but a defect in VWF activity with respect to platelet or collagen binding. We characterized novel VWF gene mutations found in type 2M VWD subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. Subjects were enrolled based on a pre-existing diagnosis of type 2M VWD. Testing included full-length gene sequencing, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding and multimer distribution. Recombinant VWF variants were synthesized using site-directed mutagenesis and expressed in HEK293T cells. Platelet binding was measured by flow cytometry with fixed platelets and ELISA with recombinant glycoprotein Ibα (GPIbα). Four novel VWF A1 domain mutations were found in individuals with type 2M VWD: S1358N, S1387I, S1394F and Q1402P. All subjects had a history of bleeding, VWF:RCo < 40 IU dL(-1) , VWF:RCo/VWF:Ag ratios <0.6 and normal multimer distribution. No defect in expression, secretion, or multimerization was found for any of the mutations. All showed decreased binding to intact platelets, and decreased or absent binding to a mutant GPIbα construct with spontaneous VWF binding. 1387I had decreased binding to all collagen types tested. 1402P had reduced binding exclusively to type VI collagen. Type 2M VWD is a heterogeneous category comprised of both collagen- and platelet-binding defects. Understanding the precise defect for each mutation may ultimately lead to better diagnosis and treatment.

Characterisation of mutations and molecular studies of type 2 von Willebrand disease

Type 2 von Willebrand disease (VWD) is characterised by qualitative defects in von Willebrand factor (VWF). Exon 28 of the VWF gene is known to be a hot spot for type 2 VWD mutations. The goal of this study was to characterise the mutations in VWF exon 28 and understand the molecular basis of phenotypes through in vitro and in silico studies. Mutation screening was performed in 56 type 2 VWD patients through direct sequencing. Expression vectors for five mutations were transiently expressed in 293-EBNA cells to understand the mutations pathology. Furthermore, in silico structure analysis was performed for 13 missense mutations. A total of 16 including eight novel mutations were detected in 23 (41%) patients. Of these, 15 were missense (including seven V1439M, A1464P, M1495L, I1509V, R1527Q, N1635I and A1647D novel ones) and one was a novel gene conversion. Expression studies and characterisation of recombinant VWF suggested the loss of VWF function for mutants P1266Q, V1439M and N1635I and gain of function for mutant R1308C. No apparent defect was seen in mutant N1231S. In silico structure analysis suggested the probable gain or loss of hydrogen/van der Waals interactions in 10 mutant proteins. In conclusion, type 2A mutations and gene conversion were found to be a common cause of type 2 VWD. Expression studies suggest the mutations N1635I for type 2A(II), P1266Q and V1439M for type 2M, R1308C for type 2B VWD and N1231S as a non-causative variant. Moreover, in silico studies of the mutants show the probable cause of respective phenotypes.

Diagnosis of von Willebrand disease in Argentina: a single institution experience

Background: von Willebrand disease (VWD) is the most common autosomal bleeding disorder, mostly inherited as dominant trait. VWD is due to deficiency/abnormality of von Willebrand factor (VWF). The true prevalence of VWD is unknown, but estimated as 0.1% to 1% of the general population. Methods: The bleeding score (BS) was used to evaluate the bleeding status of patients presenting at our institution. Laboratory analyses include: VWF:Ag, VWF:RCo, VWF:CB, multimeric pattern, VWF propeptide, Desmopressin challenge test. Genotypic analysis comprises the study of specific exons of VWF, depending on the suspected variant. Results: We describe the main characteristics of our VWD population, including phenotypic and genotypic methods used to achieve the diagnosis. From our 2,482 patients registered, quantitative variants account for 83.2% of cases, and qualitative, 16.8% of cases. Platelet-type VWD (PT-VWD) was diagnosed in two patients and acquired von Willebrand syndrome (AVWS) in six. From quantitative variants, relative frequencies are: possible type 1, 78.7%; type 1, 14.9%; type 1S, 1.5%; type 1C, 0.4%; type 3, 1.6%. From qualitative variants: 2M, 21.9%; 2B, 10.3%; 2A, 8.4%; 2N, 3.4%; patients currently without complete diagnosis: 56%. Family history of bleeding: 64.7% of cases. Type 3, 2A and 2B VWD showed the highest major bleeding (MB) frequency. Genotypic diagnosis was reached in eight type 1C, 72 type 2M, 38 type 2B, 30 type 2A, 14 type 2N, and two PT-VWD patients. Five de novo mutations were identified among 52 families (9.61%): two mutations were associated with type 2B VWD (p.V1316M and p.S1310F), one mutation associated with type 1C (Vicenza) (p.R1205H) described in combination with p.R924Q, one deletion associated to type 1 VWD according to the ISTH VWF database (p.Pro1648fs*45) in combination with p.R1426C resulting in a 2M phenotype and one mutation related to type 2A VWD (p.Y1542D). Six patients are under prophylactic treatment due to their severe bleeding: four are type 3 VWD, one type 2M VWD, and one, the patient with p.Pro1648fs*45 and p.R1426C. Conclusions: The wide heterogeneity of clinical symptoms and laboratory data from possible type 1 VWD implies a risk for bleeding that becomes crucial in challenging situations. In our population, type 2M was found to be more frequent than the type 2A variant. DDAVP (1-deamino-8-D-arginine vasopressin) challenge test, VWF propeptide/antigen ratio and genotypic studies are useful tools for discriminating cases representing difficult diagnoses such as type 2M versus type 1C VWD. Elevated propeptide ratio, the detection of antiphospholipid antibodies, platelet-associated antibodies or anti-VWF immunoglobulins and negative genotypic screening is useful to discriminate inherited VWD from AVWS. Genotypic testing assisted discrimination between types 2B versus platelet-type-VWD and type 2N VWD versus mild hemophilia A.

Cellular and molecular basis of Von Willebrand disease: Studies on blood outgrowth endothelial cells

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

The Co-Influence of VWD Type 2B and Type 2M Mutations and Platelet GPIbα On the Susceptibility of VWF to ADAMTS13 Under Shear Stress

Abstract 1108 Introduction:Von Willebrand Factor (VWF) is one kind of vital proteins involved in thrombosis, which exists in the form of multimeric glycoprotein in plasma of human. The size of VWF multimer is down regulated by metalloproteinase ADAMTS13. This physiological process is modulated by multiple factors. It was reported that the binding of platelets or GPIbα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to recombinant A1A2A3 segment in the condition of static state. Due to the mutations, VWF synthesized by patients of von Willebrand disease (VWD) type 2B and type 2M may enhance and decrease the binding of platelet membrane glycoprotein Ib (GPIb), respectively. This study aims to observe the influence of 2B/2M VWD mutations and recombinant GPIbα active fragment on the susceptibility of VWF to ADAMTS13, in the condition of high shear stress. Materials and Methods:We constructed and expressed different forms of recombinant full-length human VWF (rhVWF) including three type 2B mutations (P1337L, H1268D, R1308C), three type 2M mutations (M740I, R1205H, D1302G) and wide-type (WT) in Hela cells. Recombinant human ADAMTS13 (rhADAMTS13) and GPIbα (H1-V289) were both from our own lab. The different types of rhVWF were characterized by multimeric analysis and ristocetin-induced platelet aggregation (RIPA). We use rhADAMTS13 to digest seven full-length rhVWF in the reaction Buffer (20 mM Tris-HCl, 150mM NaCl, pH7.4, 1% bovine serum albumin) in the absence and presence of GPIbα (H1-V289). To develop a more physiological assay under fluid shear stress, we assessed the proteolytic cleavage of rhVWF by rhADAMTS13 under constant vortexing (2,500 rpm) in a PCR tube mini-mixer that mimics arterial fluid shear stress (30 dynes/cm2). The 176kDa fragment resulting from the cleavage of rhVWF at the Y1605-M1606 bond by rhADAMTS13 was visualized by 5% SDS-PAGE under reducing conditions and Western blot analysis. Results:SDS-agarose gel electrophoresis showed that all rhVWF mutants were in the similar multimeric distribution to the rhVWF-WT. In the RIPA assay, type 2B rhVWF exhibited clearly enhanced RIPA responsiveness with ristocetin at 0.5 mg/ml (R1308C > P1337L > H1268D > WT), while type2M rhVWF showed decreased RIPA responsiveness with ristocetin at 1.0 mg/ml (WT > M740I > R1205H > D1302G). At physiological salt concentration (150nM NaCl), type 2B mutants exhibited more susceptibility to rhADAMTS13 than WT and type 2M mutants in the absence of GPIbα (H1-V289). In the presence of GPIbα (H1-V289), the higher susceptibility of mutants to rhADAMTS13 was detected, which decreased in the following order: type 2B > WT > type 2M, whereas no obvious enhancement happened to the two type 2M mutants (R1205H and D1302G). Conclusions:Our study further confirmed that platelet GPIbα binding to VWF A1 domain could increase the susceptibility to ADAMTS13 at physiological conditions under high stress. The three type 2B mutations are all localized in the A1 domain and these mutations alone may also increase the susceptibility to ADAMTS13. While type 2M mutations M740I, R1205H and D1302G are localized in the D2, D3 and A1 domain, respectively. It is concluded that type 2M mutations localized close to or in the A1 domain may exhibit significantly lower susceptibility to ADAMTS13 in the presence of GPIbα (H1-V289). It may partially give the explanation that some VWD type 2M may display larger than normal VWF multimers in plasma. Disclosures:No relevant conflicts of interest to declare.

A New Microfluidic-Based Platelet Rolling Assay to Better Characterize VWD Phenotypes

Abstract 3386 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. The purpose of this study is to determine whether the degree of type 1 VWD mutant subunit incorporation into VWF multimers affects quantitative as well as qualitative defects.While one of the major functions of VWF is to capture platelets from flowing blood at the site of an injury, most clinical diagnostic assays of the platelet glycoprotein (GP) Ibα-VWF interaction are performed under static conditions, either in the presence of ristocetin or with a gain-of-function GPIbα construct. Platelet function analyzer-100 (PFA-100®) closure time is used to measure primary hemostasis in the presence of shear stress and is sensitive to low plasma VWF levels, but it can be influenced by variables such as hematocrit and platelet count.The lifetime of the GPIbα-VWF bond is prolonged with increasing shear stress to a certain threshold (catch bond), thus comprehensively studying this interaction requires using a range of shear stresses [Coburn et al, Biophys J, 2011]. We have developed an assay to examine platelet rolling on whole, recombinant (r)VWF in a microfluidic chamber. Methods:HEK293T cells were co-transfected with equal amounts of mutant VWF in a vector that appends a MycHis (mH) tag to the C-terminus, and untagged WT VWF. R1374C, a type 2M VWD mutation which interferes with the binding of VWF A1 domain to GPIbα, and R782Q, a type 1 VWD mutation, were studied here. rVWF expression was measured with ELISA, multimer composition was analyzed using SDS-agarose gel electrophoresis and Western blot, and intracellular location of rVWF was determined by immunofluorescent staining of VWF and organelle markers. FVIII binding of rVWF was performed using the Chromogenix Coatest SP4 FVIII assay.In order to study platelet-VWF interactions, recombinant VWF (10 μg/mL) from supernatants was concentrated then incubated over a surface of anti-VWF mAb, AvW-1, which we used to determine rVWF concentrations in our ELISA. Washed human platelets (6 × 108/mL) were perfused across the rVWF surface at shear stresses of 2–50 dyn/cm2 and movies were captured at 15 frames/sec. Platelet rolling velocities were manually determined during the first second that cells interacted with the surface. Differences in rolling velocity were considered significant for p<0.05 using the Mann-Whitney test. Results:Analysis of platelet rolling velocities on rVWF demonstrated a catch-slip behavior of the platelet-VWF interaction: on all rVWF surfaces, platelet rolling velocities decreased with increasing shear stress until approximately 10 dyn/cm2, above which rolling velocities increased with further increases of shear stress. Minimal platelet rolling was observed in the presence of the GPIbα-blocking antibody, AK2, or on AvW-1 surfaces incubated with concentrated supernatants from mock transfections.Compared to platelet rolling on our control surface, WT mH/WT VWF, rolling velocities on R1374C mH/WT VWF were significantly higher at shear stresses of 2, 5 and 50 dyn/cm2.R782Q mH/WT VWF exhibited pseudo Weibel-Palade body formation in HEK 293 cells, supported full multimerization and bound FVIII similarly to WT VWF. Platelet rolling velocities were significantly higher compared to WT mH/WT VWF at 50 dyn/cm2, but statistically similar at lower shear stresses. Discussion:Our microfluidic-based platelet rolling assay is able to demonstrate the catch-slip nature of the platelet GPIbα-VWF interaction. We observed distinct differences in platelet rolling velocities between a heterozygous type 2M mutant and WT VWF at a variety of shear stresses. Additionally, while individuals with the type 1 VWD mutation, R782Q, have a VWF:RCo/VWF:Ag ratio of close to 1 [Zhang et al, Hum Genet, 1995], our studies may identify a possible platelet binding defect for this mutation at high shear stresses. Thus studying the platelet-VWF interaction at a range of shear stresses may add to the completeness of VWD classification, especially in cases where VWF:RCo/VWF:Ag is close to 1. Further studies using a range of mutant:WT co-transfection ratios for type 1 VWD mutants may elucidate a role for the degree of mutant subunit incorporation into multimers in the hemostatic potential of VWF. Disclosures:No relevant conflicts of interest to declare.

New developments in the diagnosis and treatment of von Willebrand disease

von Willebrand disease (VWD) arises from deficiencies and/or defects in the plasma protein VWF. VWD is reportedly the most common inherited bleeding disorder. The classification scheme for VWD identifies six types, with type 1 VWD defining quantitative deficiencies of VWF, type 3 defining an absence of plasma VWF, and types 2A, 2B, 2M and 2N defining qualitative defects of VWF. Diagnosis requires a clinical evaluation, with evidence of both familial and personal history of (primarily mucocutaneous) bleeding, as well as laboratory assessment using a wide panel of tests, including several activity-based assays. Management of VWD typically entails standard therapy, employing desmopressin wherever possible, factor concentrates in other situations, and antifibrinolytic and additional therapies as required. Some management strategies are modified according to geographic locality. There are several developments in both the diagnosis and management of VWD that may influence the future landscape.

Characterisation of von Willebrand factor A1 domain mutants I1416N and I1416T: correlation of clinical phenotype with flow‐based platelet adhesion

Type 2M von Willebrand disease (VWD) results from mutations in the A1 domain of von Willebrand factor (VWF) that reduce its platelet-binding function. However, currently employed VWF functional static assays may not distinguish between clinical phenotype. Fifteen individuals from five kindreds with VWF-A1 domain mutations I1416T or I1416N, correlated with mild and moderate clinical phenotypes, respectively, were investigated. The mutations were reproduced by site-directed mutagenesis and expressed in HEK293T cells; functional studies of the recombinant mutants, including GPIbα binding using a flow-based assay, were performed. Plasma from all individuals demonstrated discordant reductions in VWF antigen and platelet-binding function in the presence of high-molecular-weight VWF multimers consistent with VWD type 2M. There was lowered expression and secretion of both mutants compared with wild type (WT) recombinant (r)VWF as well as a significant reduction in GPIbα binding. Binding to collagen was normal and electrophoretic analysis demonstrated a similar multimer distribution between the mutant proteins and wt-rVWF. GPIbα binding under flow was also significantly reduced for I1416N and I1416T rVWF. Impairment of GPIbα binding was more marked for I1416N rVWF than I1416T under both static and flow conditions: this was in spite of similar VWF:Ristocetin cofactor (RCo) activities in patient plasma and is consistent with a respective clinical phenotype. Our findings have established for the first time that I1416N and I1416T are responsible for a type 2M VWD phenotype and demonstrate that quantification of VWF function under shear stress may provide a more accurate measure of clinical severity than the static functional measurements in current diagnostic use.

Von Willebrand disease biology

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients

Type 2A and 2M von Willebrand disease (VWD2A and VWD2M) are characterized by the presence of a dysfunctional von Willebrand factor (VWF) and a variable bleeding tendency. So far, a head-to-head comparison of the clinical history and bleeding risk between VWD2A and VWD2M has never been provided in a prospective manner. We assessed the bleeding incidence rate and clinical characteristics in two cohorts of 17 families (46 patients) with VWD2A and 15 families (61 patients) with VWD2M prospectively followed-up for 24 months. VWF gene mutations were characterized in all of them. Mean bleeding score (BS) and VWF antigen at enrollment were significantly higher in VWD2A patients (P = 0.007). No correlation between VWF activity or factor VIII levels and the severity of BS was observed. The incidence rate of spontaneous bleeding requiring treatment was 107/100 patient-years (95% CI, 88.3-131) in VWD2A compared with 40/100 patient-years (95% CI, 30-53) in VWD2M (P < 0.001). The risk of bleeding was significantly higher in patients with BS ≥ 10 at enrollment compared with those with BS 0-2. Furthermore, 54 episodes of gastrointestinal bleeding occurred in 17/46 (36.9%) VWD2A patients and seven in 2/61 (3.3%) VWD2M patients (P < 0.0001). Bleeding tendency in VWD2A is greater than that of VWD2M, is not explained by factor VIII or VWF levels and is mainly due to an increased incidence of gastrointestinal bleeding.

Status of muscle signaling pathways in the early pathogenesis of dystroglycanopathy‐type muscular dystrophy

A subset of muscular dystrophies are caused by abnormal glycosylation of α‐dystroglycan. Without its proper sugar structure, α‐dystroglycan binding to extracellular matrix proteins (e.g. laminin, perlecan) is compromised, disrupting the structural integrity of muscle and other tissues. Mutations in the FCMD gene, encoding fukutin, cause severe and milder dystroglycanopathies, Fukuyama congenital muscular dystrophy (FCMD) and limb‐girdle muscular dystrophy type 2M (LGMD 2M), respectively. Using fukutin conditional knockout mice, we have evidence that severe congenital muscular dystrophy phenotypes involve abnormal skeletal muscle development and/or differentiation. We will present a molecular, biochemical and histological approach, screening select muscle signaling pathways in young knockout mice, to identify abnormalities in skeletal muscle development/differentiation. These findings bring new insight to potential mechanisms underlying early disease pathogenesis and severe phenotypic outcomes in dystroglycanopathy‐type congenital muscular dystrophies.This work has been supported by the Muscular Dystrophy Association (Development Grant) and the University of Georgia, College of Pharmacy.

THE PAPWORTH PLUG – successful use of high dose fibrinogen concentrate and platelet concentrate in potential life-threatening complication after cardiopulmonary bypass surgery in a patient with Type 2M Vicenza von Willebrand Disease

Anecdotally, fibrinogen concentrate (FC) has been used as a "universal" haemostatic agent in complex post-cardiopulmonary bypass (CPB) coagulopathy. We present a case where FC and two pools of platelets prevented life-threatening bleeding in a patient with moderate von Willebrand Disease (vWD) immediately post CPB.

Development of an ELISA method for testing VWF ristocetin cofactor activity with improved sensitivity and reliability in the diagnosis of von Willebrand disease

Current methods in assessing von Willebrand factor (VWF) ristocetin cofactor activity for Von Willebrand's disease (VWD) diagnosis include platelet agglutination by aggregometer or macroscopic slide examination, which are both time-consuming with suboptimal interassay and intra-assay variation. The purpose of this study is to establish a sensitive assay to detect VWF:RCo activity and evaluate its performance in VWD diagnosis. We have established a sensitive VWF:RCo-ELISA method using a monoclonal antibody, SZ-151, to immobilize the recombinant fragment of platelet glycoprotein Ib (rfGPIbα). VWF was captured by rfGPIbα in the presence of ristocetin, and then detected by HRP-conjugated rabbit anti-human VWF IgG. We tested the VWF:RCo level by this VWF:RCo-ELISA in 25 patients with different types of VWD and 36 healthy donors, and compared this method to a previously reported ELISA using 2D4 coating antibody. The sensitivity of VWF:RCo-ELISA was greatly improved with this assay (0.008 IU/dL compared to 0.031 IU/dL by 2D4 antibody). The interassay and intra-assay coefficient variation were 8% and 12%, respectively. The mean values (ranges) of VWF:RCo in patients with type 1, type 2A, type 2B, type 2M, and type 3 of VWD and control group are 31.8 (22.3-56.9), 4.8 (0.6-11.8), 8.6 (1.6-19.7), 3.9 (1.0-6.8), 1.0 (0.5-1.6), and 91.5 (47.3-169.2) IU/dL, respectively. The corresponding ratios (ranges) of VWF:RCo/VWF:Ag are 0.83 (0.70-1.16), 0.27 (0.08-0.58), 0.31 (0.15-0.40), 0.18 (0.14-0.21), 0.52 (0.13-1.19), and 0.92 (0.62-1.26). The VWF:RCo-ELISA using monoclonal anti-rfGPIbα antibody SZ-151 showed improved sensitivity and reliability in detecting VWF:RCo activity, and its clinical application would facilitate the diagnosis and classification of VWD.

Single-Center Experience of von Willebrand Disease (vWD) Among Patients with Menorrhagia: A Diagnosis which could be Missed

Menorrhagia is one of the gynecological complaints, seen in women of reproductive age. In majority of cases no organic pathology is found. To date there is no consensus on application of von Willebrand disease (vWD) testing as part of the routine investigations in menorrhagia. Diagnosis of vWD is challenging. It is complicated by intra-individual variations in von Willebrand antigen, activity, and factor VIII levels due to fluctuation of these factor levels during the menstrual cycle or hormonal therapy. The aim of this study is to detect vWD presenting with menorrhagia among Malays attending gynecology clinic by using a standard panel of haemostatic profiles. Thirty Malay patients attending gynecology clinic with unexplained menorrhagia were included in this study. Haemostatic profile such as platelet count, prothrombin time, activated partial thromboplastin time (APTT), factor VIII assay, von Willebrand factor antigen, and von Willebrand factor activity, and collagen binding assay were measured in all patients. Pre- and post hormonal haemostatic profiles were also performed in the patients diagnosed as vWD. All patients had normal APTT. Based on von Willebrand factor work-up, vWD was diagnosed in four patients (13.3%). Three of them were Type 1 and the other one was Type 2M. Investigation for vWD is essential in patients with menorrhagia and thus the laboratories performing vWD testing should provide a complete panel of diagnostic work-up in order to reduce the interpretation error. Screening for vWD should be performed before hormonal treatment as haemostatic profile post treatment could mask the diagnosis.

Hemostatic Outcomes of Hip and Knee Joint Replacement Surgery in Von Willebrand Disease (VWD) Patients: A Single Center Study

Abstract▪2279▪This icon denotes a clinically relevant abstractSurgery for patients with von Willebrand disease (VWD) is increasingly common, however there is a paucity of data on hemostatic outcomes for VWD patients undergoing joint replacement surgery when compared to patients without bleeding disorders. The aim of this study was to evaluate hemostatic prophylaxis and outcomes in VWD patients who underwent hip or knee joint replacement, with comparison to matched case control patients. Methods: We retrospectively reviewed medical records of VWD patients who underwent total hip (THA) or total knee (TKA) arthroplasty between February 1993 and January 2011 at Mayo Clinic Rochester. Outcomes for each VWD patient were compared with two case controls matched for age, gender, surgeon and surgery. For VWD patients the hemostatic prophylaxis regimen during and following surgical procedures was evaluated including results of serial monitoring of plasma levels of VWF:RCo, VWF:Ag and FVIII:C. We compared VWD and control cases for bleeding complications (major and minor), transfusion requirements, DVT (deep vein thrombosis) prophylaxis and occurrence, duration of hospitalization, and other complications. Results: Twelve VWD patients (7 female) underwent 12 TKA and 7 THA surgeries; the median age was 62 (range 42–76) years. Seven had Type 1 VWD, one had Type 3 and four had Types 2A, 2B or 2M VWD. The 19 surgical episodes were matched to 38 control surgeries. Indications for arthroplasties were mainly degenerative joint disease in both subject groups. The median BMI was 30 (range 21–45) in the VWD patients and 31.9 (20–53) in controls. Three patients with mild Type 1 VWD received during 5 surgeries only one preoperative dose of DDAVP (desmopressin 0.3μg/kg body weight). Infusions of VWF/FVIII concentrates (Humate-P or occasionally Koate-DVI) were prescribed for twelve procedures, with median preoperative dose 52 IU/kg (range 19–71). Postoperative maintenance doses were infused once or twice daily usually for up to 7 days (more than 7 days only for one Type 3 and one Type 2A VWD patient), with median ranges 22 to 38 IU/kg, and highest dose 90 IU/kg and lowest dose 11 IU/kg (typically when treatment was administrated twice daily). These substantial ranges may be explained by dose adjustments based on monitoring postoperative plasma levels of VWF:RCo and FVIII:C. The median number of infusions per procedure was 5.5 (range 1–13). One hour after the preoperative dose the median of all factors was more than 110 IU/dL, specifically VWF:RCo 154 UI/dL (range 72–277), VWF:Ag 178 IU/dL (range 101–307), and FVIII:C 114 IU/dL (range 64–290). The lowest factor levels measured during daily postoperative follow-up are represented in Figure 1. Assessment of red blood cell transfusion requirements showed no difference between baseline and D1 blood hemoglobin levels for VWD and control groups (decline of 3.0 g/dL). During follow-up, hemoglobin levels and total blood losses were also not different between both groups. However, the number of patients transfused was twice as high for the VWD group (63% vs 32% for controls, respectively), and the mean number of RBC units transfused was higher in VWD patients than in controls (1.5 vs. 0.6 units, respectively). DVT prophylaxis (low molecular weight heparin (LMWH) injections or warfarin), was administrated to 58% of VWD patients vs. 97% of controls. No postoperative thrombotic events were identified in either group. Finally, the median duration of hospitalization for VWD patients and controls was not different: 5 days (range 3–13) and 5 days (range 3–7) respectively. Conclusions: Few data are available in published literature analyzing treatment regimens and hemostatic outcomes for patients with VWD undergoing THA or TKA, a relatively common treatment for advanced degenerative joint disease. This is the first large retrospective study which reports the effectiveness of prophylactic regimens specifically in joint replacement for patients with VWD. Our results support that, as recommended in the 2008 NHLBI/NIH treatment guidelines, monitoring of plasma VWF and FVIII levels at least once daily allows for efficient and relatively low dose maintenance administration. Further, our study helps identify an adequate duration of treatment for these major surgeries, without substantively increased blood loss for VWD patients compared with controls. Disclosures:No relevant conflicts of interest to declare.

Comparative Evaluation of Five Different Assays for Ristocetin Cofactor Activity Determination,

Abstract 3317 Introduction:Despite significant advances in the comprehension of the molecular pathogenesis of Von Willebrand Disease (VWD), the diagnosis of this condition remains one of the greatest challenges of daily hematology practice, especially in settings of low prevalence such as primary care services. Ristocetin cofactor activity (VWF:RCo) is one of the pillars of the diagnosis of VWD and is also essential for its management. However, reproducibility of tests available to measure VWF:RCo is still a major issue, as evidenced by coefficient of variations (CV) as high as 30%, 45% and 27% in the ECAT, NEQAS and PALQ external quality assessment program. Classical methods to measure VWF:RCo include light-transmission platelet agregometry (LPA) and visual agglutination with formaldehyde fixed human platelet (VA), but alternative methods continue to evolve. An automated method (BC) has been recently shown superior to LPA and VA. Other methods for which limited comparative precision studies are lacking include a new LIA-based automated assay (LIA) and flow citometry (FC), using fluorescence-marked platelets and ristocetin. Methods:Here we evaluate five different VWF:RCo assays, including classical LPA and VA, two different commercially available automated coagulometer-based methods (BC and LIA), and FC. Reference ranges for the each method were established in twenty healthy adults. Plasma samples from thirty-three patients with VWD (type 1: n=26; type 2A or 2M: n=4; type 3: n=3) followed at our comprehensive care center (IHTC-Campinas, Brazil) were assayed for the comparative studies. Diagnosis of VWD was based on historical levels of VWF:Ag (assayed by ELISA) and VWF:RCo (assayed by both LTA and VA) obtained from medical records, and from bleeding history (evaluated by VWD bleeding score). Imprecision studies (intra-assay variability), were performed in different samples: commercial lyophilized normal and abnormal plasma, as well as plasma from two additional VWD patients with borderline results. All analyses were performed in the same samples, in an external quality assessment program-participating university laboratory. Results:Despite using similar conditions than those described in an original report, and repeated experiments, the flow cytometry-based assay (FC) was not reproducible in our hands, yielding extremely high CV that prevented its use in a clinical laboratory. Mean and reference ranges for VWF: RCo assays levels in healthy adults were: VA: 0.84 IU/ml (0.62–1.07); LTA: 1.13 IU/ml (0.92–1.35); BC: 0.93 IU/ml (0.75–1.12). The automated BC method yielded the lower intra-assay variability, in the range of 1.13% to 5.46% in different samples, compared to values as high as 29.4% for LTA. Correlation coefficient (r) ranged from 0.93 between BC reagent and LTA to 0.85 between VA and BC reagent. Mean VWF: RCo levels in patients ranged from 22.99 ± 17.80 with BC to 22.60 ± 18.50 with LTA and 20.11± 14.50 with VA. In twenty-six type I VWD patients, mean VWF:RCo/VW:Ag ratio ranged from was 0.7 (range 0.4–1.5) with the VA method to 0.8 (range 0.8–1.3) with the BC method. In four type 2 VWD patients, mean VWF:RCo/VW:Ag ratio was 0.2 (range 0.3–0.4) using VA method to 0.4 (range 0.4–0.5) with the BC method. Concordance, evaluated using the Bland-Altman plot, was higher between LTA and BC method. Conclusion:In our study, LTA and VA performance was consistent with historical data, presenting very high CV, but good correlation and concordance with new automated methods. Of note, VA, a simple, inexpensive and equipment-independent method, presented better CV than LTA and should be regarded as a good alternative the diagnosis of VWD in areas with limited resources. Unfortunately, flow cytometry-based VWF:RCo methods apparently suffer from the same problems of LTA and VA regarding standardization. New automated assay present enhanced precision compared to VA and LTA, with the added benefits of automation in the setting of a Hemostasis laboratory. Ongoing improvements in protocols of these new assays, leading to even higher precision, could translate into more confident limits of normal and pathological VWF:RCo levels, enabling critical changes in the diagnosis, management and even in the definition of VWD. Disclosures:No relevant conflicts of interest to declare.