Pamela A. Christopherson

Normal range of bleeding scores for the ISTH-BAT: adult and pediatric data from the merging project.

Bleeding Assessment Tools (BATs) have been developed to aid in the standardized evaluation of bleeding symptoms. The Vicenza Bleeding Questionnaire (BQ), published in 2005, established a common framework and scoring key that has undergone subsequent modification over the years, culminating in the publication of the ISTH‐BAT in 2010. Understanding the normal range of bleeding scores is critical when assessing the utility of a BAT. Within the context of The Merging Project, a bioinformatics system was created to facilitate the merging of legacy data derived from four different (but all Vicenza‐based) BATs; the MCMDM1‐VWD BQ, the Condensed MCMDM‐1VWD BQ, the Pediatric Bleeding Questionnaire and the ISTH‐BAT. Data from 1040 normal adults and 328 children were included in the final analysis, which showed that the normal range is 0–3 for adult males, 0–5 for adult females and 0–2 in children for both males and females. Therefore, the cut‐off for a positive or abnormal BS is ≥4 in adult males, ≥6 in adult females and ≥3 in children. This information can now be used to objectively assess bleeding symptoms as normal or abnormal in future studies.

Basic calcium phosphate crystals activate human osteoarthritic synovial fibroblasts and induce matrix metalloproteinase-13 (collagenase-3) in adult porcine articular chondrocytes

OBJECTIVE To determine the ability of basic calcium phosphate (BCP) crystals to induce (a) mitogenesis, matrix metalloproteinase (MMP)-1, and MMP-13 in human osteoarthritic synovial fibroblasts (HOAS) and (b) MMP-13 in cultured porcine articular chondrocytes. METHODS Mitogenesis of HOAS was measured by [3H]thymidine incorporation assay and counts of cells in monolayer culture. MMP messenger RNA (mRNA) accumulation was determined either by northern blot analysis or reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA from chondrocytes or HOAS treated with BCP crystals. MMP-13 secretion was identified by immunoprecipitation and MMP-1 secretion by western blot of conditioned media. RESULTS BCP crystals caused a 4.5-fold increase in [3H]thymidine incorporation by HOAS within 20 hours compared with untreated control cultures (p⩽0.05). BCP crystals induced MMP-13 mRNA accumulation and MMP-13 protein secretion by articular chondrocytes. In contrast, in HOAS, MMP-13 mRNA induced by BCP crystals was detectable only by RT-PCR, and MMP-13 protein was undetectable. BCP crystals induced MMP-1 mRNA accumulation and MMP-1 protein secretion by HOAS. MMP-1 expression was further augmented when HOAS were co-incubated with either BCP and tumour necrosis factor α (TNFα; threefold) or BCP and interleukin 1α (IL1α; twofold). CONCLUSION These data confirm the ability of BCP crystals to activate HOAS, leading to the induction of mitogenesis and MMP-1 production. MMP-13 production in response to BCP crystals is substantially more detectable in porcine articular chondrocytes than in HOAS. These data support the active role of BCP crystals in osteoarthritis and suggest that BCP crystals act synergistically with IL1α and TNFα to promote MMP production and subsequent joint degeneration.

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.

Calcium hydroxyapatite promotes mitogenesis and matrix metalloproteinase expression in human breast cancer cell lines

Radiographic mammary microcalcifications are one of the most pertinent diagnostic markers of breast cancer. Breast tissue calcification in the form of calcium hydroxyapatite (HA) is strongly associated with malignant disease. We tested the hypothesis that calcium HA may exert biological effects on surrounding cells, thereby facilitating breast cancer progression. Our findings showed that HA crystals enhanced mitogenesis in breast cancer cell lines MCF‐7 and Hs578T and also in normal human mammary epithelial cells. HA crystals were also found to upregulate the production of a variety of matrix metalloproteinases (MMPs), including MMP‐2, ‐9, and ‐13 in MCF‐7 and MMP‐9 in human mammary epithelial cell lines. HA crystals were found to greatly augment prostaglandin E2 levels in Hs578T cells, and treatment with a cyclooxygenase inhibitor, aspirin, abrogated the HA‐induced mitogenesis. These results suggest that calcium HA crystals may play an active role in amplifying the pathological process involved in breast cancer.

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.

The Von Willebrand Factor Propeptide (VWFpp) Traffics an Unrelated Protein to Storage

The von Willebrand factor (VWF) propeptide (VWFpp) is critical for the targeting of VWF multimers to storage granules. VWFpp alone efficiently navigates the storage pathway in AtT-20 and endothelial cells and chaperones mature VWF multimers to storage granules when the two proteins are expressed in cis or in trans. To further define the role of VWFpp in granular sorting, we examined its ability to sort an unrelated protein, C3&agr; into the regulated secretory pathway. Chimeric constructs of VWFpp and the &agr;-chain of C3 were developed. The C3&agr; protein expressed alone did not sort to granules in AtT-20 cells. The trans expression of C3&agr; and VWFpp resulted in granular storage of VWFpp but no corresponding storage of C3&agr;. When C3&agr; is expressed as a single chain molecule with VWFpp that was rendered uncleavable by furin, C3&agr; is re-routed to storage and is colocalized with VWFpp. The uncleavable protein was expressed in bovine aortic endothelial cells where it sorted to Weibel-Palade bodies, colocalized with bovine VWF, and was released when agonist stimulated. We now demonstrate that VWFpp re-routes a constitutively secreted protein to the regulated storage pathway. Furthermore, our studies suggest that the VWFpp storage signal is contained within amino acids 201 to 741.

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.

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.