P. Vincent Jenkins

Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay

Summary. This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen‐binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (≤u20030·7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (>u20030·7), but the VWF:RCo/VWF:CBA ratios were discordant (≤u20030·7) (Pu2003=u20030·001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high‐molecular‐weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet‐binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.

Survival of von Willebrand factor released following DDAVP in a type 1 von Willebrand disease cohort: Influence of glycosylation, proteolysis and gene mutations

Reduced plasma survival of von Willebrand factor (VWF) may contribute towards the pathogenesis of type 1 von Willebrand disease (VWD). However, little is known about mechanism(s) of VWF clearance and factors that may affect it. The half-life of VWF-related parameters following the administration of DDAVP was measured in 26 patients with type 1 VWD and 10 haemophilia A controls. Binding of lectins Ricinus communis (RCA-I) and Erythina crystagalli (ECA) agglutinins to VWF and VWF susceptibility to ADAMTS-13-mediated proteolysis were investigated. Sequence analysis of targeted regions of the VWF gene was performed to inspect for mutations that have been associated with increased clearance. Post-DDAVP clearance of VWF was increased approximately three-fold in the type 1 VWD cohort overall. However this was not shown to consistently associate with steady-state VWF antigen (VWF:Ag) levels. Furthermore, increased VWF clearance was not consistently associated with increased ratios of VWF propeptide (VWFpp) to VWF:Ag indicating that a normal ratio does not necessarily reflect normal post-DDAVP survival in type 1 VWD patients. RCA-I and ECA binding to VWF were increased in type 1 VWD patients and, although inversely correlated with VWF levels, this was independent of VWF clearance. There was no association between VWF clearance and ADAMTS-13-mediated proteolysis. Three novel candidate mutations with an increased clearance phenotype were identified. The data are consistent with heterogeneity in pathogenic mechanisms in type 1 VWD and are consistent with type 1 VWD representing a complex genetic trait.

Screening for Candidate Mutations Causing von Willebrand’s Disease (vWD)

von Willebrand factor (vWF) is a large, complex glycoprotein that exists in plasma and platelets, and is synthesized by megakaryocytes and endothelial cells. vWF plays an essential role in hemostasis in at least two ways. It is involved in platelet adhesion to the damaged vascular endothelium and also stabilizes factor VIII in plasma by acting as its carrier molecule. vWF has a multidomain structure, composed of multiples of four domain types A-D in the arrangement D1-D2-D-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2 (1). After initial synthesis, the protein is modified by post-translational processing of dimerization and multimerization and cleavage of the D1-D2 domain of the propeptide. The monomeric subunit of 220 kDa forms multimers of a range up to 20mDA. The different functions of vWF are assigned to different domains. The A1 domain is involved in binding of vWF to platelet glycoprotein 1b (GpIb), binding to fibrillar collagen, sulfatides, and heparin. The D3 domain is involved in the binding of factor VIII, while the binding of the platelet GpIIb-IIIa receptor is localized to the carboxy-terminal end of the C2 domain. A collagen binding site important in binding to collagen type III is present in the A3 domain.

Multiplex PCR for Detection of the Prothrombin 3′-UTR (G20210A) Polymorphism and the Factor V Leiden Mutation

Prothrombotic evaluation of patients with a history-and in particular a family history-of venous thromboembolic disease is becoming increasingly important as our understanding of the molecular abnormalities that underlie this clinical disorder increases. A recently described G→A polymorphism at position 20210 in the 3-untranslated region of the prothrombin gene (F2 3-UTR) has been found to be associated with an increased risk of venous thrombotic disease. In the Leiden Thrombophilia Study (LETS), the prevalence of carriers of the 20210 A allele in the healthy population was 2.3%, among patients with a single objectively proven DVT 6.2% and in a selected group of patients with a personal and family history of venous thrombosis 18%.

Detection of mutations causing hemophilia a using an in vitro coupled transcription and translation system.

Mutation detection in the factor VIII gene is complicated by the size and complexity of the gene-186 kb spanning 26 exons. The exons vary in size from 69 bp to 3106 bp and the introns from 207 bp to 32.4 kb (1). The first mutations to be identified in the factor VIII gene involved mutations at Taq I restriction sites (an enzyme that contains the mutational CpG dinucleotide within its recognition sequence) or were large deletions detected by Southern blotting (2). Small deletions, substitution, and missense mutations proved more difficult to detect as these appear to be randomly distributed throughout the factor VIII gene. For these reasons, therefore, many laboratories involved in carrier detection in Hemophilia A have used an indirect procedure known as gene tracking or linkage analysis. This involves the use of various polymorphic markers (RFLPs and VNTRs) to follow the segregation of the defective factor VIII gene through individuals in a family (This is not covered in this chapter, but an excellent review on the subject is available [3]). Several factors limit this technique, primarily the need for intervening family members, the need for a proband to be present, the occasional need for paternity testing, and the frequent occasions where all polymorphic markers prove to be uninformative. Furthermore, it adds little to our understanding of the mutations that underlie Hemophilia A.

Inversion Mutation Analysis in Hemophilia A by Restriction Enzyme Analysis and Southern Blotting

Intron 22 of the factor VIII gene contains a 9.5kb region of DNA that is repeated on at least two other locations telomeric to, and at least 500 kb from, the gene. These regions are termed intron 22 homologous regions (int22h) (1) and contain the Factor VIII associated gene (F8A).