Von Willebrand Factor

July 19, 2002
14:00 to 18:00
Georgian Room
Boston Park Plaza Hotel

Chairman:  R. Montgomery, USA
Co-chairs:  G. Castaman, Italy; J. Eikenboom, The Netherlands; A. B. Federici, Italy;
A. Goodeve, UK; P. A. Kouides, USA; C. Mazurier, France; J. H. Rand, USA

The VWF Subcommittee was attended by about 125 attendees at the first session and 100 participants for the second session.  They actively participated in the discussion of the various talks provided by the program.  The following represents a full summary of the program.

Cooperative Studies (J. Eikenboom, Chair)

Dr. Goodeve reported on the European study "Molecular and Clinical Markers for Diagnosis and Management of type 1 von Willebrand Disease" funded by the European Union. Twelve centers from 9 European countries cooperate in this study. The aim of this study is to determine the value of clinical, phenotypic and molecular markers for the diagnosis of type 1 VWD; to examine the contribution of polymorphism and mutation in the VWF gene to type 1 VWD; and to determine the proportion of type 1 VWD not associated with mutation in VWF gene. Two hundred families with VWD type 1 and 1200 healthy controls will be included. So far 142 families have been recruited. In each family data is collected on bleeding history. Basic laboratory tests for VWF parameters and advanced phenotypic tests (VWF:CB, VWF:FVIIIB, platelet VWF) will be performed. Linkage analysis will be performed to establish or reject co-segregation between the phenotype and VWF genotype and to establish the contribution of the VWF gene to the variability in VWF level. The influence of haplotype (5 SNPs) on plasma VWF level will be studied. Finally, mutation analysis will be performed in all families and mutations will be expressed in vitro. To date, mutations have been identified in 24 index cases and no mutation was identified in 2 cases.

Dr. Lillicrap reported on the Canadian study "Molecular Genetic Basis of Type 1 von Willebrand Disease" funded by the Canadian Institutes of Health Research. Seven clinics have recruited 172 individuals from 52 families. To date 29 families were analyzed. For genotype analysis they were all screened for seven polymorphic sites and two type 1 dominant negative mutations (C1130F and C1149R). Phenotypic analysis showed a slight excess of women among the affected individuals. Blood group O was over-represented among index cases. Overall phenotypic data from the source laboratories agree with the core laboratory, although there were some discrepancies.
 
Dr. Rodeghiero reported the final results of the "Validation of the Diagnostic Criteria of Type 1 von Willebrand Disease: an International Multicenter Study" that was initiated by the SSC in 1997. The aim of this study was to investigate the contribution of bleeding history to the diagnosis in a sample of obligatory carriers of type 1 (n=42) and type 3 VWD (n=70). Bleeding history was compared with that of affected members and age and sex-matched controls (n=224). A standardized questionnaire was used to evaluate each hemorrhagic symptom at presentation, using a score system (0: no symptoms, 1: mild symptoms; 2: symptoms requiring medical attention only; 3: symptoms requiring hospitalization, replacement therapy or blood transfusion). The severity of epistaxis, menorrhagia and bleeding after tooth extraction is similar but not equal in obligatory carriers of type 3 VWD and controls; affected subjects in type 1 and 3 families show a clear prevalence of score 1, 2 and 3. For type 1 VWD obligatory carriers vs. controls, menorrhagia and epistaxis have a low sensitivity, whereas cutaneous bleeding is the most sensitive symptom. For type 3 VWD obligatory carriers vs. controls, menorrhagia and bleeding after tooth extraction have a low sensitivity, whereas bleeding after surgery is the most sensitive symptom. Laboratory screening in subjects with at least two hemorrhagic symptoms seems a reasonable option.

Database and Classification (A. Goodeve, Chair)

New Molecular Database.  Anne Goodeve summarized the new ISTH VWF website, on behalf of Stuart Croft (Sheffield) and Ross MacLachlan (Kingston), who have designed the site. It is intended that this site will replace the previous VWF site, hosted at the University of Michigan. The web address is http://www.shef.ac.uk/vwf/ .

The aim of the web site is to provide a convenient, user-friendly first-point of reference for the scientific community with an interest in von Willebrand factor and von Willebrand disease.  The site contains searchable mutation and polymorphism data, the full human VWF genomic sequence plus links to several other VWF sequences, diagrams of VWF and links to other sites of interest.

Linkage analysis in the EU study.  Jeroen Eikenboom described that three short tandem repeat polymorphisms in the VWF gene will be used to examine linkage to the gene in families with type 1 VWD. Analysis will be performed using software such as LINKAGE and MLINK. In addition to highlighting families with VWD not linked to the VWF gene, the contribution of the gene to the variability in VWF level will be determined.

VWD Classification Issues (J.E. Sadler, Chair)

Strengths and weaknesses of the current VWD classification system were discussed. Dr. Sadler suggested that a classification should be designed with a primary emphasis on clinical utility. Thus, disease categories should correlate with bleeding risk and response to specific therapies and should be assignable using common laboratory tests. Although the current classification satisfies these guidelines, some patients are difficult to classify. In particular, decreased VWF antigen and proportionally decreased VWF activity may be consistent with VWD type 1, but circulating VWF may contain subunits with mutations that do not affect binding functions. These patients could be accommodated under VWD type 1 by including variants in which circulating VWF contained mutant subunits but otherwise had a normal proportion of large multimers with apparently normal function.

Dr Castaman presented data on VWD Vicenza, another difficult to classify variant. The consensus opinion was that it was premature to define this as a distinct variant. Some data is being generated on what features are related to specific mutations within the VWF gene in these patients.

Dr. Eikenboom presented the results of an extensive comparison between patients with a diagnosis of VWD type 1 and obligate carriers of VWD type 3 alleles.

Dr. Montgomery discussed the heterogeneity of mutations and mechanisms underlying the categories of VWD types 1, 2A and 2M.

Dr. Budde proposed an extended classification in which VWD type 2A would be split into subtypes based mainly on multimer structure and secondarily on mutation location. After discussion, the consensus opinion was that all such mutations converge on two classes of mechanism: impaired multimer assembly and increased catabolism of large multimers. All such patients lack circulating large multimers, which impairs hemostasis, and they generally have a poor response to DDAVP. Phenotypic variation within any of the proposed subcategories appears to exceed the variation observed between them. Splitting VWD type 2A therefore was not felt to have added clinical utility.

Dr. Budde presented the results of studies using multimer analysis to distinguish VWD type 1 from VWD type 2 variants.

Dr. Eikenboom presented results on mutations that affect collagen binding specifically. These currently would be included under VWD type 2M because they impair platelet adhesion.

While a number of issues were brought up, it was decided to continue the present classification system and to extend the 2A classification to incorporate the specific genetic mutation and perhaps a multimer description as a parenthetic remark.  No working party was developed at this time.

Nomenclature (G. Castaman, Chair)

Dr. Mazurier summarized the current recommended abbreviations for VWF activities as published in Thrombosis and Haemostasis in 1991.  All authors, reviewers, and Editors were requested to use this nomenclature.  

Dr. Mazurier proposed an abbreviation for VWF multimers as VWF:Mult or VWF:Mers.  Since these were not quantitative measurements, it was decided to refer to these as VWF multimers rather than any further abbreviation.  A group will be appointed to make recommendations by next year on methods to quantitate the relative concentration of the various size multimers.

Dr. Montgomery made two proposals.  The first was to cease using the term VW AgII to refer to the VWF propeptide.  Although VWFpp or VWF:pp were proposed as abbreviations, there was no consensus for a change.  The first recommendation to stop using VW AgII was accepted.  The second recommendation to use abbreviations for the propeptide was not accepted at this time.  In publications the propeptide should be referred to as the VWF propeptide.  

Standardization Issues (A. Federici, Chair)

Dr. Federici presented the interim report of the Working Party to study the standardization of VWD diagnosis.  A working party and time-lines were presented, and it is expected to have an interim report by the next meeting in Birmingham in 2003.  Thirty centers will be involved in the study and at least 1/3 of them will be in developing countries.

Anthony Hubbard presented data on the collagen binding of the VWF concentrate standard.  It was recommended that there needs to be further work on this matter and that a small working group will study the use of different collagen binding assays in several assays using several techniques in each participating laboratory and will make a report at the next SSC Meeting in 2003.

The issue of standardization of the collagen-binding assay was discussed by Drs. Mazurier, Sietz, and Lillicrap.  There continue to be difficulties with agreeing on a standard approach to this assay.  Dr. Federici agreed to head a working party to address this problem and to report on this at the next SSC meeting.

Dr. Mazurier will head a working party to recommend standards reporting of quantitative assessment of multimer size.  Dr. Budde presented data on the graphical approach and this needs to be extended to other laboratories and countries.

Treatment Issues (P. Kouides and R. Montgomery, Co-Chairs)

A working group headed by Dr. Federici will report at the next SSC meeting on treatment and monitoring of patients using DDAVP.  Dr. Thompson summarized the recovery and half-lives of various treatment concentrates.  Dr. Mannucci presented the results of his survey on thrombosis in patients treated with VWF concentrate.  Seven additional patients were identified through this survey.  This brought up a problem with our network for the VWF committee since we previously had no method of disseminating information about a potential problem (thrombosis) with VWF treatment concentrates.  We are attempting to develop an e-mail list for the future.  Further information was provided on FVIII and VWF levels in patients and treaters are encouraged to monitor FVIII levels.

VWF-cleaving protease (ADAMTS-13) (J. Rand, Chair)

Dr. Pier Mannucio Mannucci reported on the results of the assay when applied to patients with diseases other than TTP. He reviewed data that were published in BLOOD last year (Sept 2002) that showed decreases of the VWF-protease levels,generally mild-moderate, in a variety of clinical conditions.

Dr Han Mou Tsai reviewed the recent identification of the VWF-cleaving protease as ADAMTS13 and reported on his assay methodology.  The protease has been purified from plasma and the gene has been mapped to chromosome 9q34. Other studies report that some patients without TTP have low (<30%) levels of the VWF-cleaving protease and suggest that the decrease may have pathophysiological implications. A review of his data reveals that mildly decreased ADAMTS13 levels are detected in some patients with various pathological conditions, however, the decreases are generally not as profound as described by others. The discrepancy most likely results from difference in the assay methods, some of which generate broad ranges among normal individuals. Since the decrease does not correlate with the severity of the underlying diseases and is not associated with an increased VWF size, the significance of the observed decrease is unclear and should be interpreted with caution. He also compared the various current assay methodologies.
 
Dr. Martina Bohm presented her and Dr. Inge Scharrer’s data on a new assay method for the protease that utilizes measurement of VWF:RCo. This test appears to have benefits in its simplicity and nonreliance upon gel-based analysis of multimers or protein fragments.

There was general agreement that laboratories currently performing these assays be encouraged to evaluate comparability by testing shared specimens. Dr. Bohm informed the group that such an effort, sponsored by Dr. Laemle, was already underway.

Final recommendations

1. The use of VW AgII should no longer be used for the propeptide of VWF.  No abbreviation should be used  - use "VWF propeptide."
2. No abbreviation for VWF multimers should be formalized – use "VWF multimers."
3. A working party was formed for assessing distribution of VWF multimer size with Dr. Mazurier as head.
4. A working party on the collagen-binding assay was formed with Dr. Federici as head.
5. A working party on treatment and monitoring of patients after DDAVP was established under the direction of Dr. Federici.
6. An informal group to exchange plasmas for ADAMTS 13 was agreed to by the major labs and was encouraged to determine if the SSC plasma standard has normal ADAMTS 13 level and if it has potential for being a future formal or informal standard.

Robert R. Montgomery, Chair