Veronica H. Flood

VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population

Diagnosis and classification of VWD is aided by molecular analysis of the VWF gene. Because VWF polymorphisms have not been fully characterized, we performed VWF laboratory testing and gene sequencing of 184 healthy controls with a negative bleeding history. The controls included 66 (35.9%) African Americans (AAs). We identified 21 new sequence variations, 13 (62%) of which occurred exclusively in AAs and 2 (G967D, T2666M) that were found in 10%-15% of the AA samples, suggesting they are polymorphisms. We identified 14 sequence variations reported previously as VWF mutations, the majority of which were type 1 mutations. These controls had VWF Ag levels within the normal range, suggesting that these sequence variations might not always reduce plasma VWF levels. Eleven mutations were found in AAs, and the frequency of M740I, H817Q, and R2185Q was 15%-18%. Ten AA controls had the 2N mutation H817Q; 1 was homozygous. The average factor VIII level in this group was 99 IU/dL, suggesting that this variation may confer little or no clinical symptoms. This study emphasizes the importance of sequencing healthy controls to understand ethnic-specific sequence variations so that asymptomatic sequence variations are not misidentified as mutations in other ethnic or racial groups.

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.

Limitations of the ristocetin cofactor assay in measurement of von Willebrand factor function.

Summary.  Background: Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in von Willebrand factor (VWF) and diagnosed by a disproportionate decrease in VWF ristocetin cofactor activity (VWF:RCo) as compared with VWF antigen (VWF:Ag). Objective: We report here on the spurious diagnosis of VWD in a patient with a sequence variation in the ristocetin‐binding domain of VWF. Patients/methods: The index case had a VWF:RCo of 11 IU dL−1, with VWF:RCo/VWF:Ag ratio of 0.09. DNA sequencing revealed a novel P1467S mutation in a known ristocetin‐binding region of the A1 domain. Because of the discrepancy between the laboratory findings, consistent with type 2M VWD, and the patient’s lack of bleeding symptoms, further studies were performed to determine whether this mutation affected VWF function or merely reduced its ability to interact with ristocetin. Results: Studies with recombinant VWF showed normal platelet binding with botrocetin, but a significant decrease in binding in response to ristocetin. Ristocetin‐induced binding to recombinant GPIb was also absent, but normal binding was seen when a gain‐of‐function GPIb construct was used in the absence of ristocetin. VWF function under shear stress was normal when analyzed with a cone and plate(let) analyzer. Conclusions: The decreased VWF:RCo seen with the P1467S sequence variation likely represents an artifact as a result of the use of ristocetin to measure VWF activity. The normal VWF function in other assays correlates with the lack of hemorrhagic symptoms, and suggests the need for more physiologically relevant assays of VWF function.

Absent Collagen Binding in a VWF A3 Domain Mutant: Utility of the VWF:CB in Diagnosis of VWD

Von Willebrand factor (VWF) tethers platelets to injured subendothelium through binding sites for collagen and platelet glycoprotein Ib (GPIb). The collagen binding site has been localized to the VWF A1 and A3 domains [1]. This interaction is measured in vitro by the VWF collagen binding assay, or VWF:CB [2,3]. VWD guidelines recently published by the NHLBI suggest restricting use of the VWF:CB to subjects who have abnormal initial screening results with VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo) [4]. The VWF:RCo assay measures VWF-platelet interactions, as induced by the antibiotic ristocetin, and therefore a defect exclusive to the VWF-collagen axis could potentially be missed by omitting the VWF:CB assay. We report here on a subject with type 1 VWD who was discovered to have abnormal collagen binding and a mutation in the VWF A3 domain. The index case and family members were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWF after informed consent was obtained. VWF:Ag, VWF:RCo, VWF:CB, multimer distribution, and blood type analysis were performed in the clinical hemostasis laboratory of the BloodCenter of Wisconsin as previously described [5]. This subject had VWF:Ag of 41 IU/dL and VWF:RCo 44 IU/dL with normal multimer distribution, but VWF:CB was only 22 U/dL (figure 1A). Her history was significant for epistaxis requiring cautery and hemorrhage following tonsillectomy as a child. She also had prolonged bleeding following a dilation and curettage procedure. Her bleeding score, calculated using the scoring system from the European MCMDM-1 VWD study, was elevated at 8 [6]. DNA sequencing of the full length VWF coding sequence, performed on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA), showed this subject was heterozygous for a mutation in exon 31 (5356C>G) which led to substitution of aspartic acid for the wild-type histidine at amino acid 1786 (H1786D). Figure 1 Panel A shows VWF levels for the H1786D proband and family members. VWF:Ag, VWF:RCo, and VWF:CB listed for each subject were performed in the clinical laboratory along with blood type and multimer analysis. The VWF:CB used type III collagen. In addition, ... Recombinant VWF containing the 1786D mutation was synthesized via site-directed mutagenesis using the Stratagene QuikChange kit (La Jolla, CA) and expressed in HEK293T cells. Additional constructs were synthesized to incorporate three previously reported mutations in the collagen binding domain, 1731T [7], 1745C, and 1783A [8]. For the research laboratory collagen binding assays, ELISA plates were coated with either type I human placental collagen (Sigma, St. Louis, MO) at 5 μg/mL or type III human placental collagen (Southern Biotech) at 1 μg/mL, diluted in carbonate coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, 3 mM sodium azide), and incubated at 4°C overnight. Either plasma or recombinant VWF diluted in phosphate-buffered saline with 1% BSA was added to each well and incubated at room temperature for 1 hour. VWF bound to collagen was detected using a biotin-conjugated polyclonal antibody to VWF (Dako, Carpinteria, CA) also diluted in phosphate-buffered saline with 1% BSA. Both the 1786D and the 1731T constructs had normal expression compared to wild-type (WT) VWD. Multimer analysis showed a full spectrum of multimers, with no decrease in high molecular weight multimers for the A3 domain mutants. Collagen binding studies were performed in our research laboratory using recombinant WT, 1786D, and 1731T VWF. Results are expressed as a ratio of VWF:CB to VWF:Ag. The previously described 1731T mutation showed a reduction in binding to both type I and type III collagen, at 45% and 50% of WT. In contrast, the 1786D mutant showed barely detectable binding to type I and type III collagen, at <1% of wild-type (figure 1B). Similar lack of binding was seen with the 1745C and 1783A constructs. The profound defect in collagen binding shown with the 1786D construct is supported by studies of the VWF A3 domain crystal structure, which shows H1786 at one of the interfaces with collagen [9]. Replacement of this histidine with alanine also abolished VWF-collagen binding [10]. The S1731T mutation previously reported by Ribba and colleagues led to a decrease in VWF:CB [7]. A recent report by Riddell and colleagues details two A3 domain mutations, W1745C and S1783A, both with a significant decrease in VWF:CB [8]. Three additional A3 domain mutations, Q1734H, I1741T, and Q1762R, have also been reported to be associated with decreased collagen binding although the affected subjects did not display profound bleeding symptoms [11]. Both the VWF A1 and A3 domains are capable of binding collagen [1]. The major binding site is thought to be in the A3 domain [12]. The in vitro collagen binding assays with 1786D VWF did not demonstrate any evidence of compensatory A1 domain binding. It is possible that in vivo the A1 domain binding site can adequately compensate for the H1786D mutation in the A3 domain when VWF is activated under flow conditions, which may decrease the symptoms seen in the patients with this defect. Previous work with recombinant H1786A VWF supports this hypothesis, as it has been demonstrated that under flow conditions, binding to collagen can occur through the VWF A1 domain [13]. It is also possible that the severity of the in vitro phenotype is partially alleviated by the presence of some normal VWF in the heterozygous patients since multimeric VWF would likely contain some monomers with the intact collagen binding site. The H1786D mutation fits best into the current VWD classification system as a type 2M variant, with abnormal VWF function despite normal multimer distribution. Not all 2M mutations, however, have abnormal collagen binding. Previous work from our laboratory demonstrated the A1 domain mutations F1369I and I1425F had normal binding to type III collagen, while an 11 amino acid deletion mutant, Δ1392–1492, did appear to affect collagen binding [14]. These patients, however, all presented with decreased VWF:RCo/VWF:Ag ratios, prompting their inclusion as 2M mutations. The H1786D mutation described here has a different effect on VWF function than those mutations considered as classic 2M. Defects in collagen binding may ultimately require an alternate classification scheme to distinguish them from platelet-binding defects. This case also suggests that the VWF:CB assay may have utility in diagnosis of variant VWD.

Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage.

Type 2A VWD is characterized by the absence of large VWF multimers and decreased platelet-binding function. Historically, type 2A variants are subdivided into group 1, which have impaired assembly and secretion of VWF multimers, or group 2, which have normal secretion of VWF multimers and increased ADAMTS13 proteolysis. Type 2A VWD patients recruited through the T. S. Zimmerman Program for the Molecular and Clinical Biology of VWD study were characterized phenotypically and potential mutations identified in the VWF D2, D3, A1, and A2 domains. We examined type 2A variants and their interaction with WT-VWF through expression studies. We assessed secretion/intracellular retention, multimerization, regulated storage, and ADAMTS13 proteolysis. Whereas some variants fit into the traditional group 1 or 2 categories, others did not fall clearly into either category. We determined that loss of Weibel-Palade body formation is associated with markedly reduced secretion. Mutations involving cysteines were likely to cause abnormalities in multimer structure but not necessarily secretion. When coexpressed with wild-type VWF, type 2A variants negatively affected one or more mechanisms important for normal VWF processing. Type 2A VWD appears to result from a complex intersection of mechanisms that include: (1) intracellular retention or degradation of VWF, (2) defective multimerization, (3) loss of regulated storage, and (4) increased proteolysis by ADAMTS13.

Critical von Willebrand factor A1 domain residues influence type VI collagen binding.

Summary.  Background:  von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen.

Crucial role for the VWF A1 domain in binding to type IV collagen

Von Willebrand factor (VWF) contains binding sites for platelets and for vascular collagens to facilitate clot formation at sites of injury. Although previous work has shown that VWF can bind type IV collagen (collagen 4), little characterization of this interaction has been performed. We examined the binding of VWF to collagen 4 in vitro and extended this characterization to a murine model of defective VWF-collagen 4 interactions. The interactions of VWF and collagen 4 were further studied using plasma samples from a large study of both healthy controls and subjects with different types of von Willebrand disease (VWD). Our results show that collagen 4 appears to bind VWF exclusively via the VWF A1 domain, and that specific sequence variations identified through VWF patient samples and through site-directed mutagenesis in the VWF A1 domain can decrease or abrogate this interaction. In addition, VWF-dependent platelet binding to collagen 4 under flow conditions requires an intact VWF A1 domain. We observed that decreased binding to collagen 4 was associated with select VWF A1 domain sequence variations in type 1 and type 2M VWD. This suggests an additional mechanism through which VWF variants may alter hemostasis.

Third Åland islands conference on von Willebrand disease, 26-28 September 2012: meeting report.

The first meeting of international specialists in the field of von Willebrand disease (VWD) was held in the Åland islands in 1998 where Erik von Willebrand had first observed a bleeding disorder in some members of a family from Föglö and a summary of the meeting was published in 1999. The second meeting was held in 2010 and a report of the meeting was published in 2012. Topics covered included progress in understanding of VWD over the last 50 years; multimers; classification of VWD; pharmacokinetics and laboratory assays; genetics; treating the paediatric patient; prophylaxis; geriatrics; gene therapy and treatment guidelines. This third meeting held over 3 days covered the structure and function of von Willebrand factor (VWF); type 1 VWD, the most common form of the disease; a lifespan of pharmacokinetics in VWD; detecting inhibitors in VWD patients; and special challenges in understanding and treating the female VWD patient.

Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease

Summary.  Background:  von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Collagen binding assays (VWF:CB), however, are not part of the routine work‐up for von Willebrand disease (VWD).

Critical VWF A1 Domain Residues Influence Type VI Collagen Binding

Summary.  Background:  von Willebrand factor (VWF) binds to subendothelial collagen at sites of vascular injury. Laboratory testing for von Willebrand disease (VWD), however, does not always include collagen binding assays (VWF:CB) and standard VWF:CB assays use type I and/or type III collagen rather than type VI collagen.