Genetic determinants of plasma von Willebrand factor antigen levels: a target gene SNP and haplotype analysis of ARIC cohort

Abstractvon Willebrand factor (VWF) is an essential component of hemostasis and has been implicated in thrombosis. Multimer size and the amount of circulating VWF are known to impact hemostatic function. We associated 78 VWF single nucleotide polymorphisms (SNPs) and haplotypes constructed from those SNPs with VWF antigen level in 7856 subjects of European descent. Among the nongenomic factors, age and body mass index contributed 4.8% and 1.6% of VWF variation, respectively. The SNP rs514659 (tags O blood type) contributed 15.4% of the variance. Among the VWF SNPs, we identified 18 SNPs that are associated with levels of VWF. The correlative SNPs are either intronic (89%) or silent exonic (11%). Although SNPs examined are distributed throughout the entire VWF gene without apparent cluster, all the positive SNPs are located in a 50-kb region. Exons in this region encode for VWF D2, D′, and D3 domains that are known to regulate VWF multimerization and storage. Mutations in the D3 domain are also associated with von Willebrand disease. Fifteen of these 18 correlative SNPs are in 2 distinct haplotype blocks. In summary, we identified a cluster of intronic VWF SNPs that associate with plasma levels of VWF, individually or additively, in a large cohort of healthy subjects.

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Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients

Blood ◽

10.1182/blood.2019000190 ◽

2019 ◽

Vol 134 (11) ◽

pp. 880-891 ◽

Cited By ~ 7

Author(s):

Laura L. Swystun ◽

Kenichi Ogiwara ◽

Orla Rawley ◽

Christine Brown ◽

Ilinca Georgescu ◽

...

Keyword(s):

Blood Group ◽

Von Willebrand Factor ◽

Half Life ◽

Hemophilia A ◽

Blood Type ◽

Abo Blood Group ◽

Genetic Determinants ◽

Group Status ◽

Von Willebrand ◽

Willebrand Factor

Abstract Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients. We observed that VWF:Ag and VWF propeptide (VWFpp)/VWF:Ag, but not VWFpp, were associated with FVIII half-life. VWFpp/VWF:Ag negatively correlated with FVIII half-life in patients with non-O blood type, but no correlation was observed for type O patients, suggesting that von Willebrand factor (VWF) half-life, as modified by the ABO blood group, is a strong regulator of FVIII PK. The FVIII-binding activity of VWF positively correlated with FVIII half-life, and the rare or low-frequency nonsynonymous VWF variants p.(Arg826Lys) and p.(Arg852Glu) were identified in patients with reduced VWF:FVIIIB but not VWF:Ag. Common variants at the VWF, CLEC4M, and STAB2 loci, which have been previously associated with plasma levels of VWF and FVIII, were associated with the FVIII PK profile. Together, these studies characterize the mechanistic basis by which VWF clearance and ABO glycosylation modify FVIII PK in a pediatric population. Moreover, this study is the first to identify non-VWF and non-ABO variants that modify FVIII PK in pediatric hemophilia A patients.

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Variation at the von Willebrand Factor (vWF) Gene Locus Is Associated With Plasma vWF:Ag Levels: Identification of Three Novel Single Nucleotide Polymorphisms in the vWF Gene Promoter

Blood ◽

10.1182/blood.v93.12.4277 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4277-4283 ◽

Cited By ~ 74

Author(s):

Angela M. Keightley ◽

Y. Miu Lam ◽

Jolene N. Brady ◽

Cherie L. Cameron ◽

David Lillicrap

Keyword(s):

Single Nucleotide Polymorphisms ◽

Von Willebrand Factor ◽

Gene Locus ◽

Strong Linkage Disequilibrium ◽

Nucleotide Polymorphisms ◽

Polymorphic Variation ◽

Single Nucleotide ◽

Haplotype 1 ◽

Von Willebrand ◽

Willebrand Factor

Abstract Both genetic and environmental factors contribute to the normal population variability of plasma von Willebrand Factor (vWF) levels, however, regulatory mechanisms at the vWF gene locus itself have not yet been identified. We have investigated the association between polymorphic variation in the 5′-regulatory region of the vWF gene and levels of plasma vWF:Ag in a study of 261 group O blood donors. Three novel single nucleotide polymorphisms (SNPs) were identified in the vWF promoter: C/T at -1234, A/G at -1185, and G/A at -1051. These SNPs had identical allele frequencies of 0.36 for the -1234C, -1185A, and -1051G alleles and 0.64 for the -1234T, -1185G, and -1051A alleles and were in strong linkage disequilibrium. In fact, these polymorphisms segregated as two distinct haplotypes: -1234C/-1185A/-1051G (haplotype 1) and -1234T/-1185G/-1051A (haplotype 2) with 12.6% of subjects homozygous for haplotype 1, 40.6% homozygous for haplotype 2, and 42.5% of subjects heterozygous for both haplotypes. Only 4.3% of individuals had other genotypes. A significant association between promoter genotype and level of plasma vWF:Ag was established (analysis of covariance [ANCOVA], P = .008; Kruskal-Wallis test,P = .006); individuals with the CC/AA/GG genotype had the highest mean vWF:Ag levels (0.962 U/mL), intermediate values of vWF:Ag (0.867 U/mL) were observed for heterozygotes (CT/AG/GA), and those with the TT/GG/AA genotype had the lowest mean plasma vWF:Ag levels (0.776 U/mL). Interestingly, when the sample was subgrouped according to age, the significant association between promoter genotype and plasma vWF:Ag level was accentuated in subjects > 40 years of age (analysis of variance [ANOVA], P = .003; Kruskal-Wallis test, P= .001), but was not maintained for subjects ≤ 40 years of age (ANOVA, P > .4; Kruskal-Wallis test, P > .4). In the former subgroup, mean levels of plasma vWF:Ag for subjects with the CC/AA/GG, CT/AG/GA, and TT/GG/AA genotypes were 1.075, 0.954, and 0.794 U/mL, respectively. By searching a transcription factor binding site profile database, these polymorphic sequences were predicted to interact with several transcription factors expressed in endothelial cells, including Sp1, GATA-2, c-Ets, and NFκB. Furthermore, the binding sites at the -1234 and -1051 SNPs appeared to indicate allelic preferences for some of these proteins. Electrophoretic mobility shift assays (EMSAs) performed with recombinant human NFκB p50 showed preferential binding of the -1234T allele (confirmed by supershift EMSAs), and EMSAs using bovine aortic endothelial cell (BAEC) nuclear extracts produced specific binding of a nuclear protein to the -1051A allele, but not the -1051G allele. These findings suggest that circulating levels of vWF:Ag may be determined, at least in part, by polymorphic variation in the promoter region of the vWF gene, and that this association may be mediated by differential binding of nuclear proteins involved in the regulation of vWF gene expression.

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Variation at the von Willebrand Factor (vWF) Gene Locus Is Associated With Plasma vWF:Ag Levels: Identification of Three Novel Single Nucleotide Polymorphisms in the vWF Gene Promoter

Blood ◽

10.1182/blood.v93.12.4277.412k03_4277_4283 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4277-4283 ◽

Cited By ~ 4

Author(s):

Angela M. Keightley ◽

Y. Miu Lam ◽

Jolene N. Brady ◽

Cherie L. Cameron ◽

David Lillicrap

Keyword(s):

Single Nucleotide Polymorphisms ◽

Von Willebrand Factor ◽

Gene Locus ◽

Strong Linkage Disequilibrium ◽

Nucleotide Polymorphisms ◽

Polymorphic Variation ◽

Single Nucleotide ◽

Haplotype 1 ◽

Von Willebrand ◽

Willebrand Factor

Both genetic and environmental factors contribute to the normal population variability of plasma von Willebrand Factor (vWF) levels, however, regulatory mechanisms at the vWF gene locus itself have not yet been identified. We have investigated the association between polymorphic variation in the 5′-regulatory region of the vWF gene and levels of plasma vWF:Ag in a study of 261 group O blood donors. Three novel single nucleotide polymorphisms (SNPs) were identified in the vWF promoter: C/T at -1234, A/G at -1185, and G/A at -1051. These SNPs had identical allele frequencies of 0.36 for the -1234C, -1185A, and -1051G alleles and 0.64 for the -1234T, -1185G, and -1051A alleles and were in strong linkage disequilibrium. In fact, these polymorphisms segregated as two distinct haplotypes: -1234C/-1185A/-1051G (haplotype 1) and -1234T/-1185G/-1051A (haplotype 2) with 12.6% of subjects homozygous for haplotype 1, 40.6% homozygous for haplotype 2, and 42.5% of subjects heterozygous for both haplotypes. Only 4.3% of individuals had other genotypes. A significant association between promoter genotype and level of plasma vWF:Ag was established (analysis of covariance [ANCOVA], P = .008; Kruskal-Wallis test,P = .006); individuals with the CC/AA/GG genotype had the highest mean vWF:Ag levels (0.962 U/mL), intermediate values of vWF:Ag (0.867 U/mL) were observed for heterozygotes (CT/AG/GA), and those with the TT/GG/AA genotype had the lowest mean plasma vWF:Ag levels (0.776 U/mL). Interestingly, when the sample was subgrouped according to age, the significant association between promoter genotype and plasma vWF:Ag level was accentuated in subjects > 40 years of age (analysis of variance [ANOVA], P = .003; Kruskal-Wallis test, P= .001), but was not maintained for subjects ≤ 40 years of age (ANOVA, P > .4; Kruskal-Wallis test, P > .4). In the former subgroup, mean levels of plasma vWF:Ag for subjects with the CC/AA/GG, CT/AG/GA, and TT/GG/AA genotypes were 1.075, 0.954, and 0.794 U/mL, respectively. By searching a transcription factor binding site profile database, these polymorphic sequences were predicted to interact with several transcription factors expressed in endothelial cells, including Sp1, GATA-2, c-Ets, and NFκB. Furthermore, the binding sites at the -1234 and -1051 SNPs appeared to indicate allelic preferences for some of these proteins. Electrophoretic mobility shift assays (EMSAs) performed with recombinant human NFκB p50 showed preferential binding of the -1234T allele (confirmed by supershift EMSAs), and EMSAs using bovine aortic endothelial cell (BAEC) nuclear extracts produced specific binding of a nuclear protein to the -1051A allele, but not the -1051G allele. These findings suggest that circulating levels of vWF:Ag may be determined, at least in part, by polymorphic variation in the promoter region of the vWF gene, and that this association may be mediated by differential binding of nuclear proteins involved in the regulation of vWF gene expression.

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Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor

Blood ◽

10.1182/blood-2009-10-249102 ◽

2010 ◽

Vol 116 (2) ◽

pp. 280-286 ◽

Cited By ~ 95

Author(s):

Veronica H. Flood ◽

Joan Cox Gill ◽

Patricia A. Morateck ◽

Pamela A. Christopherson ◽

Kenneth D. Friedman ◽

...

Keyword(s):

African American ◽

Von Willebrand Disease ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Functional Defect ◽

Hemorrhagic Risk ◽

Clinical Biology ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor

Abstract The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of “VWF activity” by this assay and may not reflect a functional defect or true hemorrhagic risk.

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The mutation N528S in the von Willebrand factor (VWF) propeptide causes defective multimerization and storage of VWF

Blood ◽

10.1182/blood-2009-09-244327 ◽

2010 ◽

Vol 115 (22) ◽

pp. 4580-4587 ◽

Cited By ~ 22

Author(s):

Sandra L. Haberichter ◽

Ulrich Budde ◽

Tobias Obser ◽

Sonja Schneppenheim ◽

Cornelia Wermes ◽

...

Keyword(s):

Von Willebrand Factor ◽

Mammalian Cells ◽

Consensus Sequence ◽

Von Willebrand Disease ◽

Relative Reduction ◽

Homozygous Mutation ◽

Storage Granules ◽

Von Willebrand ◽

And Storage ◽

Willebrand Factor

Abstract We characterized a consanguineous Turkish family suffering from von Willebrand disease (VWD) with significant mucocutaneous and joint bleeding. The relative reduction of large plasma von Willebrand factor (VWF) multimers and the absent VWF triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective α-granular storage of larger multimers. Patients were nearly unresponsive to desmopressin acetate, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage also in endothelial cells. We identified an N528S homozygous mutation in the VWF propeptide D2 domain, predicting the introduction of an additional N-glycosylation site at amino acid 526 in close vicinity to a “CGLC” disulphide isomerase consensus sequence. Expression studies in mammalian cells demonstrated that N528S-VWF was neither normally multimerized nor trafficked to storage granules. However, propeptide containing the N528S mutation trafficked normally to storage granules. Our data indicate that the patients' phenotype is the result of defective multimerization, storage, and secretion. In addition, we have identified a potentially novel pathogenic mechanism of VWD, namely a transportation and storage defect of mature VWF due to defective interaction with its transporter, the mutant propeptide.

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Cosegregation of von Willebrand factor gene polymorphisms and possible germinal mosaicism in type IIB von Willebrand disease

Blood ◽

10.1182/blood.v77.7.1476.1476 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1476-1483 ◽

Cited By ~ 8

Author(s):

EW Murray ◽

AR Giles ◽

PJ Bridge ◽

IR Peake ◽

DP Lillicrap

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Nucleotide Substitutions ◽

Gene Encoding ◽

Single Nucleotide ◽

Unaffected Parent ◽

Von Willebrand ◽

Willebrand Factor ◽

Germinal Mosaicism ◽

Significant Linkage

Abstract Recent reports of the mutations resulting in von Willebrand disease (vWD) have indicated that some cases of type IIA vWD are caused by single nucleotide substitutions in the gene encoding von Willebrand factor (vWF). However, the molecular pathogenesis of type IIB vWD remains unresolved and, with the complex posttranslational processing required for fully functional vWF, the mutations responsible for this phenotype may occur at loci other than the vWF gene. This study has used six intragenic vWF polymorphisms to assess the linkage of type IIB vWD to this gene in three families (48 individuals). The results of these studies indicate that there is significant linkage between the vWF gene and the type IIB phenotype (logarithm of the odds ratio of 7.2 at theta = 0), suggesting that the mutations responsible for this disorder frequently occur at this locus. Results from one of these families indicates that the disorder has been transmitted from an unaffected parent to two children who have inherited the same vWF gene as seven unaffected siblings. This finding is suggestive of the presence of germinal mosaicism for the mutation in the father.

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Cosegregation of von Willebrand factor gene polymorphisms and possible germinal mosaicism in type IIB von Willebrand disease

Blood ◽

10.1182/blood.v77.7.1476.bloodjournal7771476 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1476-1483

Author(s):

EW Murray ◽

AR Giles ◽

PJ Bridge ◽

IR Peake ◽

DP Lillicrap

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Nucleotide Substitutions ◽

Gene Encoding ◽

Single Nucleotide ◽

Unaffected Parent ◽

Von Willebrand ◽

Willebrand Factor ◽

Germinal Mosaicism ◽

Significant Linkage

Recent reports of the mutations resulting in von Willebrand disease (vWD) have indicated that some cases of type IIA vWD are caused by single nucleotide substitutions in the gene encoding von Willebrand factor (vWF). However, the molecular pathogenesis of type IIB vWD remains unresolved and, with the complex posttranslational processing required for fully functional vWF, the mutations responsible for this phenotype may occur at loci other than the vWF gene. This study has used six intragenic vWF polymorphisms to assess the linkage of type IIB vWD to this gene in three families (48 individuals). The results of these studies indicate that there is significant linkage between the vWF gene and the type IIB phenotype (logarithm of the odds ratio of 7.2 at theta = 0), suggesting that the mutations responsible for this disorder frequently occur at this locus. Results from one of these families indicates that the disorder has been transmitted from an unaffected parent to two children who have inherited the same vWF gene as seven unaffected siblings. This finding is suggestive of the presence of germinal mosaicism for the mutation in the father.

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Influence of single nucleotide polymorphisms in factor VIII and von Willebrand factor genes on plasma factor VIII activity: the ARIC Study

Blood ◽

10.1182/blood-2011-10-383661 ◽

2012 ◽

Vol 119 (8) ◽

pp. 1929-1934 ◽

Cited By ~ 17

Author(s):

Marco Campos ◽

Ashley Buchanan ◽

Fuli Yu ◽

Maja Barbalic ◽

Yang Xiao ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Factor Viii ◽

Von Willebrand Factor ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Fviii Activity ◽

Von Willebrand ◽

Aric Study ◽

Willebrand Factor ◽

Intronic Snps

AbstractFactor VIII (FVIII) functions as a cofactor for factor IXa in the contact coagulation pathway and circulates in a protective complex with von Willebrand factor (VWF). Plasma FVIII activity is strongly influenced by environmental and genetic factors through VWF-dependent and -independent mechanisms. Single nucleotide polymorphisms (SNPs) of the coding and promoter sequence in the FVIII gene have been extensively studied for effects on FVIII synthesis, secretion, and activity, but impacts of non–disease-causing intronic SNPs remain largely unknown. We analyzed FVIII SNPs and FVIII activity in 10 434 healthy Americans of European (EA) or African (AA) descent in the Atherosclerosis Risk in Communities (ARIC) study. Among covariates, age, race, diabetes, and ABO contributed 2.2%, 3.5%, 4%, and 10.7% to FVIII intersubject variation, respectively. Four intronic FVIII SNPs associated with FVIII activity and 8 with FVIII-VWF ratio in a sex- and race-dependent manner. The FVIII haplotypes AT and GCTTTT also associated with FVIII activity. Seven VWF SNPs were associated with FVIII activity in EA subjects, but no FVIII SNPs were associated with VWF Ag. These data demonstrate that intronic SNPs could directly or indirectly influence intersubject variation of FVIII activity. Further investigation may reveal novel mechanisms of regulating FVIII expression and activity.

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The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646298 ◽

1992 ◽

Vol 68 (04) ◽

pp. 464-469 ◽

Cited By ~ 9

Author(s):

Y Fujimura ◽

S Miyata ◽

S Nishida ◽

S Miura ◽

M Kaneda ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Von Willebrand Factor ◽

Binding Activity ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Normal Individuals ◽

Rag 1 ◽

The One ◽

Von Willebrand ◽

Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

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Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649724 ◽

1993 ◽

Vol 70 (06) ◽

pp. 1053-1057 ◽

Cited By ~ 32

Author(s):

Agnès M Journet ◽

Simin Saffaripour ◽

Denisa D Wagner

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Von Willebrand Factor ◽

Deletion Mutants ◽

Relative Importance ◽

D2 Domain ◽

Constitutive Secretion ◽

Von Willebrand ◽

And Storage ◽

Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

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