Luminographic Detection of von Willebrand Factor Multimers in Agarose Gels and on Nitrocellulose Membranes

Budde, Ulrich; Schneppenheim, R.; Plendl, Hansjörg; Ruggeri, Zaverio M.; Zimmerman, Theodore S.

doi:10.1055/s-0038-1645215

Cited by 118 publications

(89 citation statements)

References 14 publications

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“…VWF multimers were analyzed by electrophoresis using a 1.6% sodium dodecyl sulfate (SDS) agarose gel followed by electrotransfer to a nylon membrane, and the multimers were visualized using the chemiluminescent visualization kit from Amersham Pharmacia Biotech (Baie D'Urfe, PQ, Canada). 22 …”

Section: Coagulation Studiesmentioning

confidence: 99%

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease

O’Brien

James

Othman

et al. 2003

Blood

121

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To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312؊19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312؊19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD. (Blood. 2003;102:549-557)

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Section: Coagulation Studiesmentioning

confidence: 99%

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease

O’Brien

James

Othman

et al. 2003

Blood

121

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“…21 Medium (1.6%) and low resolution (1.2%) gels (LGT agarose type VII; Sigma, Munich, Germany) were prepared in running gel buffer (0.375 mol/L Tris, 0.1% SDS, pH 8.8) and cast in a 21.5 ϫ 12.5 cm ϫ 1.5 mm thick frame. After solidifying, a 21.5 ϫ 2.5 cm ϫ 1.5 mm strip of 0.8% agarose stacking gel [HGT (P) agarose; Marine Colloids, Rockland, ME] prepared in stacking gel buffer (0.125 mol/L Tris, 0,1% SDS, pH 6.8) was poured.…”

Section: Plasma Vwf Multimer Analysismentioning

confidence: 99%

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

Suva

Hartman

Dilley

et al. 2008

The American Journal of Pathology

110

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The platelet glycoprotein Ib-IX receptor binds surfacebound von Willebrand factor and supports platelet adhesion to damaged vascular surfaces. A limited number of mutations within the glycoprotein Ib-IX complex have been described that permit a structurally altered receptor to interact with soluble von Willebrand factor, and this is the molecular basis of platelet-type von Willebrand disease. We have developed and characterized a mouse model of platelet-type von Willebrand disease (G233V) and have confirmed a platelet phenotype mimicking the human disorder. The mice have a dramatic increase in splenic megakaryocytes and splenomegaly. Recent studies have demonstrated that hematopoetic cells can influence the differentiation of osteogenic cells. Thus, we examined the skeletal phenotype of mice expressing the G233V variant complex. At 6 months of age, G233V mice exhibit a high bone mass phenotype with an approximate doubling of trabecular bone volume in both the tibia and femur. Serum measures of bone resorption were significantly decreased in G233V animals. With decreased bone resorption, cortical thickness was increased, medullary area decreased, and consequently, the mechanical strength of the femur was significantly increased. Using ex vivo bone marrow cultures, osteoclast-specific staining in the G233V mutant marrow was diminished, whereas osteoblastogenesis was unaffected. These studies provide new insights into the relationship between the regulation of megakaryocytopoiesis and bone mass. (Am J

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“…Therefore, for a case with a VWF:RCo < 0.10 IU mL -1 we used 0.10 IU mL -1 as the absolute value, and we recognize that we may have overestimated the true VWF:RCo level and the RCo/Ag ratio in some cases. FVIII assays were performed according to published methods [22], as were VWF multimer analyses [23]. The VWF multimers were reviewed by three independent observers, and rated for the presence/absence of HMW multimers and triplet structure.…”

Section: Coagulation Studiesmentioning

confidence: 99%

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

Notley

Hegadorn

et al. 2007

Journal of Thrombosis and Haemostasis

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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, chisquared 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 genotypephenotype 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.

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Luminographic Detection of von Willebrand Factor Multimers in Agarose Gels and on Nitrocellulose Membranes

Cited by 118 publications

References 14 publications

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

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

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