Colleen Notley

The C-type lectin receptor CLEC4M binds, internalizes and clears von Willebrand factor and contributes to the variation in plasma von Willebrand factor levels

Genetic variation in or near the C-type lectin domain family 4 member M (CLEC4M) has been associated with plasma levels of von Willebrand factor (VWF) in healthy individuals. CLEC4M is a lectin receptor with a polymorphic extracellular neck region possessing a variable number of tandem repeats (VNTR). A total of 491 participants (318 patients with type 1 von Willebrand disease [VWD] and 173 unaffected family members) were genotyped for the CLEC4M VNTR polymorphism. Family-based association analysis on kindreds with type 1 VWD demonstrated an excess transmission of VNTR 6 to unaffected individuals (P = .0096) and an association of this allele with increased VWF:RCo (P = .029). CLEC4M-Fc bound to VWF. Immunofluorescence and enzyme-linked immunosorbent assay demonstrated that HEK 293 cells transfected with CLEC4M bound and internalized VWF. Cells expressing 4 or 9 copies of the CLEC4M neck region VNTR showed reduced interaction with VWF relative to CLEC4M with 7 VNTR (CLEC4M 4%-60% reduction, P < .001; CLEC4M 9%-45% reduction, P = .006). Mice expressing CLEC4M after hydrodynamic liver transfer have a 46% decrease in plasma levels of VWF (P = .0094). CLEC4M binds to and internalizes VWF, and polymorphisms in the CLEC4M gene contribute to variable plasma levels of VWF.

Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study.

Summary.  Background: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. Aim: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. Method: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. Results and conclusion: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family‐based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO ‘O’ and ‘A’ alleles and the VWF codon 1584 variant. There was also weak association with the −1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

In vivo evaluation of an adenoviral vector encoding canine factor VIII : High-level, sustained expression in hemophiliac mice

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Frequency of Platelet type versus Type 2B von Willebrand Disease: An international registry-based study

Less than 50 patients are reported with platelet type von Willebrand disease (PT-VWD) worldwide. Several reports have discussed the diagnostic challenge of this disease versus the closely similar disorder type 2B VWD. However, no systematic study has evaluated this dilemma globally. Over three years, a total of 110 samples/data from eight countries were analysed. A molecular approach was utilised, analysing exon 28 of the von Willebrand factor (VWF) gene, and in mutation negative cases the platelet GP1BA gene. Our results show that 48 cases initially diagnosed as putative type 2B/PT-VWD carried exon 28 mutations consistent with type 2B VWD, 17 carried GP1BA mutations consistent with a PT-VWD diagnosis, three had other VWD types (2A and 2M) and five expressed three non-previously published exon 28 mutations. Excluding 10 unaffected family members and one acquired VWD, 26 cases did not have mutations in either genes. Based on our study, the percentage of type 2B VWD diagnosis is 44% while the percentage of misdiagnosis of PT-VWD is 15%. This is the first large international study to investigate the occurrence of PT-VWD and type 2B VWD worldwide and to evaluate DNA analysis as a diagnostic tool for a large cohort of patients. The study highlights the diagnostic limitations due to unavailability/poor application of RIPA and related tests in some centres and proposes genetic analysis as a suitable tool for the discrimination of the two disorders worldwide. Cases that are negative for both VWF and GP1BA gene mutations require further evaluation for alternative diagnoses.

The effect of exercise on von Willebrand factor and ADAMTS‐13 in individuals with type 1 and type 2B von Willebrand disease

Summary.  Background: The effect of exercise on von Willebrand factor (VWF) and ADAMTS‐13 levels in individuals with von Willebrand disease (VWD) has never been reported.

Challenges in defining type 2M von Willebrand disease: results from a Canadian cohort study

Summary.  Background/methods: In order to better characterize the genotype–phenotype correlation in type 2M von Willebrand disease (VWD), we sequenced the coding region for the mature subunit of the von Willebrand factor (VWF) gene (exons 18–52, including exon/intron boundaries) in 16 index cases originally submitted to the Canadian Type 1 VWD Study as type 1 VWD, but reclassified as type 2M VWD on the basis of phenotype (excessive mucocutaneous bleeding and von Willebrand factor: antigen (VWF:Ag) and/or von Willebrand factor: ristocetin cofactor (VWF:RCo) between 0.05 and 0.50 IU mL–1 on at least two occasions and RCo/Ag ratio < 0.6 and no loss of high molecular weight multimers). Available family members (16 affected, 23 unaffected and six unknown) were sequenced for identified mutations. Results: We identified eight different missense mutations (R854Q, T1054M, R1315C, R1374C, R1374H, L1382P, S2179F, and T2647M) within these 16 families. We were significantly more likely to identify a VWF mutation in cases with RCo/Ag ratios < 0.50 (P < 0.05, chi‐squared test). Importantly, every index case with an RCo/Ag ratio < 0.40 (4/4 index cases) had a mutation identified within the A1 domain, in contrast to 1/12 cases with an RCo/Ag ratio > 0.40. Difficulties with the standardization of the VWF:RCo may be responsible for the heterogeneity in cases with RCo/Ag ratios between 0.40 and 0.60. Conclusions: The genotype–phenotype correlation for cases with RCo/Ag ratios < 0.40 is clear. On the basis of our results, the phenotypic definition of type 2M VWD may need to be more stringent, and should be the subject of an international standardization initiative.

The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles.

Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion.

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models

Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamic-expressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant ADAMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model.

Influence of a GT repeat element on shear stress responsiveness of the VWF gene promoter

Summary.  Background: Plasma von Willebrand factor (VWF) is mainly derived from endothelial cells, cells that express a large repertoire of genes that are transcriptionally regulated by fluid shear stress. Endothelial VWF expression is not uniform throughout the vasculature, and levels are increased at regions associated with disturbed blood flow and steep gradients of shear stress. It is, however, unknown whether shear stress influences the regulation of VWF gene expression. Objectives: Our objective was to evaluate the effect of shear stress on endogenous endothelial VWF mRNA expression and VWF promoter (− 2722 to − 1224) activity and to determine whether genetic elements modulate this flow‐induced expression. Methods: A parallel plate flow chamber was used to expose endothelial cells to a shear level of 15 dynes cm−2 for 24 or 6 h. VWF mRNA expression was analyzed. Various VWF promoter constructs that each contain either SNP haplotypes 1 or 2 and either a 17‐GT or a 23‐GT repeat element were transfected into endothelial cells, and flow‐induced promoter activation was assessed. Results: When endothelial cells were exposed to shear stress, endogenous VWF mRNA expression increased 1.84‐fold and average VWF promoter activity was enhanced 3.4‐fold. Single nucleotide polymorphisms at − 2708 and − 2525, and the shear stress‐response element at − 1585, are not responsible for the shear stress‐induced increase. Rather a GT repeat element at − 2124 mediates the increase in activity, and the length of this polymorphic repeat element influences the magnitude of induction. Conclusions: Shear stress enhances VWF promoter activity and a polymorphic GT repeat element mediates the stress‐induced transactivation.

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor.

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605‐M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13‐mediated cleavage of VWF. Recombinant human full‐length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1·5 mol/l urea, and analyzed via multimer migration distance. The glutathione‐S‐transferase (GST) and histidine‐tagged construct, E1554‐R1668 of VWF (VWF115) was assayed via enzyme‐linked immunosorbent assay: VWF115 was bound to anti‐GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase‐labelled anti‐histidine tag antibody. All alterations examined in the Y1605‐M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A‐domain changes in VWF alter ADAMTS13‐mediated proteolysis.