Von Willebrand Factor
August 6th , 2005
11:00 to 14:30
Ballroom 2
Sydney Convention & Exhibition Centre, Sydney
Chairman: A. B. Federici, Italy
Co-chairs: G. Castaman, Italy; J Di Paola, USA; J. Eikenboom, The Netherlands; E. Favaloro, Australia; A. Goodeve, UK; D. Lillicrap, Canada; C. Mazurier, France;
R. Montgomery, USA; R. Schneppenheim, Germany
Summary of Approvals and Working Parties:
A) Progress reports on different Working Parties
(Chairs: J. Eikenboom and E. Favaloro)
The session started with a report on VWF assays in VWD diagnosis by A. Hubbard & C.A. Lee. The objective of the working party is to determine the best diagnostic repertoire for VWD diagnosis and thereby produce guidelines for the minimal requirements for correct VWD diagnosis. This will be achieved through a review of methods currently in use and a multi-center international study in which plasma samples covering a range of VWD variants will be dispatched for diagnosis. Trial fills on VWD plasma have indicated that lyophilisation does not affect the estimation of VWF. A panel of plasma samples from patients with known VWF mutations (and viral marker negative) were lyophilised in 2004. Thirty four laboratories from 17 different countries have agreed to participate and dispatch of the coded samples is almost complete. Participants are requested to return information on their methodologies as well as laboratory data to support their diagnosis of the lyophilised samples. Analysis of the results is planned for the last quarter of 2005.
L. De Marco & E. Favaloro reported on the results of the WP on different collagen reagents. It is intended that the VWF:CB working party undertake a study to help evaluate the utility of different collagen preparations in the diagnosis of VWD, and in the discrimination of qualitative VWD Types, as well as for identification of functionality of VWF in therapeutic factor replacement products. The VWF:CB study will entail (i) retesting of an identical plasma set by a smaller select number of expert laboratories using methodologies currently existing in those laboratories as well as by additional methodologies, including commercial options, (ii) additional testing of in-house well characterised plasma material with all methods, (iii) testing of therapeutic VWF concentrates. Companies producing commercial VWF:CB kits have agreed to participate and different collagen preparations will also be evaluated. Proposed members and participants of this exercise will be Dr Favaloro, Dr De Marco, Dr Hubbard, Dr. Federici & Dr Mazurier, plus other interested parties to be identified. It is proposed that this study commences after completion of the above mentioned diagnostics study. In the interim, the members of the VWF:CB working party will formulate a study protocol that identifies inclusions and requirements.
U. Budde & C. Mazurier reported on the progress of the WP on multimeric analysis. Since the session in Venice we had requests from 3 more laboratories to analyse our samples. Only 1 of these did send results and one laboratory analysed their results in a way compatible to the others. This brings up seven results that can be evaluated statistically for most of the 10 samples. While for the 4 samples with a full or near full content of large multimers the coefficient of variation for the content of >10 multimers was acceptable (below 15%), it was much too high for those samples that missed greater parts of the large multimers: between 52% and 81%. Thus the method is still far from standardized. We await results from a WP of VWF assays (results from 30 laboratories) and from the type 1 study before proceeding further in our aim to standardize the multimeric analysis.
The WP on VWD classification has produced a report with recommendations for VWD classification, which was presented by J.E. Sadler. As of the SSC meeting in Venice in 2004, the Working Party on the classification of VWD had evaluated recent published advances in our understanding of the pathophysiology of VWD and made significant progress in revising the classification to reflect this progress. Substantial changes were made to the definition of VWD and of VWD type 1. At this time, a preliminary manuscript for the revised classification has been written and extensively revised. Before it is submitted for publication, the Working Party would prefer to include the major findings of the Canadian and European studies of VWD type 1, which will be relevant to the changes proposed for VWD type 1. The first papers from these studies should be submitted for publication with the next few months. During the next year, the working party will review the published results of Canadian and European studies of VWD type 1, and other ongoing studies of VWF assays and multimer analysis. With the benefit of these data, a manuscript entitled "Update on the classification of von Willebrand disease" will be submitted for publication no later than the next SSC meeting in 2006.
The session ended with a report by L. Hilbert & D. Lillicrap on a new WP on VWF molecular biology and expression studies. The aim of this working party is to complete a review on the methods used for the identification and expression of molecular abnormalities in VWD. A questionnaire was sent to laboratories involved in two parallel projects on type 1 VWD: the European MCMDM-1VWD project and the Canadian project. The major conclusions from this pilot survey were presented and indicated that there is some consensus in the methods used for the identification of a VWF gene abnormalities and for the construction of mutated expression vectors harbouring a VWD mutation. However, transient transfections were performed using different procedures, in different cell types and this results in recombinant VWF antigen levels that may vary by a 10-fold factor according to the laboratory. In order to conduct a second larger survey, this questionnaire will be sent to other laboratories willing to participate. Those interested should send an e-mail to hilbert@lfb.fr or lillicrap@cliff.path.queenu.ca .
B) Progress reports on ADAMTS13 assays & clinical applications
(Chairs, Anne Goodeve & Reinhard Schneppenheim)
R. de Cristoforo reported on the kinetics of VWF cleavage by ADAMTS13 at different concentrations of NaCl. Low concentrations of NaCl significantly enhance cleavage of VWF compared to physiologic NaCl concentrations. Testing of different kations and anions, respectively, revealed that the inhibitory effect of higher salt concentrations was dependent on anions rather than on cations. It was suggested that the inhibitory effect was due to conformational changes of VWF that could be reversed by ristocetin. Accordingly, anions should stabilize a more closed structure of the VWF A2 domain, whereas shear should stabilize an open form.
H. Kokame reported on normal values for ADAMTS13 of a large Japanese control population of 3,822 individuals obtained by the commercially available ADAMTS13 FRET assay developed by the group. They observed a slow decrease of fluorescence at zero activity. The lower limit of detection was estimated as 3 % of normal, and the assay was able to differentiate between values of 3 %, 0 % and 5 %. The minimum value was 43 %, and the reference range was 52 – 172 %.
M. Matsumoto reported on a new ELISA based on MoABs directed against the N-terminal decapeptide that is exposed after ADAMTS13 digest of VWF. This assay is very sensitive and specific with a detection limit of 0.5 % which would be superior to all other assays to date. The correlation with classical assays like the multimer method was good.
J. Kremer-Hovinga reported on her experience measuring the titer of autoantibodies against ADAMTS13 in 74 patients with TTP/HUS by a new ELISA (Technozyme ADAMTS13 Inh, Technoclone, Vienna ). The group detected antibodies in 93 % of cases with ADAMTS13 activity < 10 %, in 39 % of cases with ADAMTS13 between 10 and 25 %, in 8 % of cases with ADAMTS13 between 26 and 50 %. The limit of detection was estimated as < 1BU which would be an improvement compared to the standard Bethesda method.
R. Schneppenheim and U. Budde showed experimental data about the action of ADAMTS13 on different VWF mutants. The group reproduced the enhanced susceptibility of classical VWD type 2A (IIA) mutants for ADAMTS13 proteolysis not only for the group 2 mutations, previously defined as being susceptible for enhanced proteolysis but also for a group 1 mutation in the A2 domain (S1506L) for which previously intracellular retention of large VWF multimers was made responsible for their observed lack. The group could also show by in vitro mutagenesis involving single and multiple substitutions of amino acids that Y1505 and M1506 flanking the VWF proteolytic site are not essential for recognition by ADAMTS13.
F. Peyvandi reported on the Italian experience of TTP in 140 patients. Plasma level of ADAMTS13 was reduced to < 10% in only 57 % of patients in acute phase. Measurements of ADAMTS13 in remission phase of recurrent patients more often revealed lower levels than in patients with a single episode. Severe ADAMTS13 deficiency (<10 %) was associated with a high prevalence of neutralizing inhibitors (61 %). 12 % of patients without measurable neutralizing antibodies had non-neutralizing antibodies on Western blotting. In about 1/3 of patients with severe ADAMTS13 deficiency no neutralizing antibodies were found which could not be explained by diagnosis of the inherited form of TTP predisposition (Upshaw-Schulman syndrome, USS) alone, since only 5/140 (3 %) patients had USS.
C) Other reports and proposal for projects and surveys
(Chairs: J. Di Paola and D. Lillicrap)
Update of the Working Party on DDAVP in VWD : Dr. Lethagen summarized the current status of this project. He introduced the project web site and re-iterated the main points of the project. In addition to the aim to enroll 150 VWD patients for the main biological response and clinical efficacy part of the study, there is also a plan to assess a detailed pharmacokinetic profile in 40 patients.
Long-term prophylaxis of VWD : Dr Abshire introduced this new activity. He described the rationale, organization, objectives and entry criteria for the proposed VWD prophylaxis project. This project will be organized by the newly created VWD Prophylaxis Network and will be sponsored by an investigator-driven grant from ZLB-Behring.
Update of the Registry on Acquired von Willebrand Syndrome : Dr. Budde presented a brief update on the activities associated with this registry. He provided details on the new Registry web site (www.intreavws.com) and the information that could be entered at this site.
Protocol on non-immune thrombocytopenia and type 2B VWD : Dr. Bussel introduced this project and discussed the details of a proposed questionnaire that would be sent to interested investigators. He encouraged anyone with an interest in this area to contact him.
US NHLBI VWD Document : Dr. Nichols presented information about the recently completed US NHLBI “clinical guidelines” project on VWD.