Von Willebrand Factor
June 29-30, 2006
Oslo Kongressenter, Norway
Chairman: A. B. Federici, Italy
Co-chairs: T. Abshire, USA; G. Castaman, Italy; J. Di Paola, USA; J. Eikenboom, The Netherlands; E. Favaloro, Australia; A. Goodeve, UK; D. Lillicrap, Canada; R. Schneppenheim, Germany
Summary of Approvals and Working Parties:
The VWF Subcommittee was attended by about 160 attendees at the first session and by about 130 at the second session. They actively participated in the discussion of the various topics provided in the program. The following represents the minutes of the program.
1) WP on VWF Assays (J. Eikenboom, The Netherlands)
Final report of the Working Party on VWF assays in VWD
The organization of the study was reported by Dr C.A. Lee. Thirty two laboratories from 17 countries have participated in a study to evaluate VWF assays in the diagnosis of von Willebrand Disease (VWD) with the objective of determining the best diagnostic repertoire for VWD. Each laboratory received 11 or 12 individually coded samples comprising 6 VWD patient samples with known genotype (types 1/2N, 2A, 2B, 2M, 2M smear, 2N) and 2 normal control samples (Group O and non-Group O) with 3 or 4 coded duplicate samples. All plasma samples were lyophilized in 0.5 ml aliquots and comparisons with frozen aliquots have indicated that the multimeric profile has not been affected by the lyophilization procedure. Laboratories were requested to perform the following 5 assay methods: VWF:Ag, VWF:RCo, VWF:CB, VWF:FVIIIB and multimeric analysis. Samples were dispatched in May/June 2005 and results were returned by November 2005. Laboratories returned details of methodology together with a diagnosis and supporting laboratory data for each sample. The results have been decoded, sorted according to sample and dispatched for analysis by expert laboratories designated for each method.
Dr K. Friedman reported on the VWF:Ag assay. A diversity of reference calibrators was used (3 rd-5 th WHO standard). There was also variation in the reference ranges used (population based or ABO blood group stratified), in the size of the population surveyed for range definition, and in the lower limit of the range. Furthermore, the use of concordance ratios is unreliable when the VWF:Ag is low.
Dr E.J. Favaloro reported on the VWF:CB assay. The VWF:CB assay is effective in identifying loss of HMW multimers associated with type 2A and 2B VWD. VWF:CB (especially type I(/III mixture) collagen based) is in this respect better than VWF:RCo. The results are, however, dependent on the source of the collagen used. VWF:RCo is better in identifying type 2M with a functional defect. VWF:CB does not replace VWF:RCo, but could possibly replace the need for multimers in selected cases.
Dr C.A. Sabin reported on the statistical aspects of the study. The diagnostic accuracy was high for 2A, 2N and normal individuals, but poor for 1/2N compound heterozygote, 2B, 2M and 2M smear individuals. The diagnostic accuracy was higher in the labs performing all assays. There was a good agreement (kappa value) between repeated diagnosis, however this is a measure of agreement and not of accuracy as consistently incorrect diagnoses give a high kappa value. Additional information was provided by Dr. Budde and Castaman on VWF Multimers and VWF:RCo assays.
VWF collagen binding versus VWF:RCo activities
Dr. A.B. Federici informed the audience that Dr. De Marco could not attend but he is still working on the isolation and characterization of human collagen type VI: he probably will report on this issue in Geneva . Dr. Federici presented data on the relationships between VWF:CB and VWF:RCo in a sample population of patients with VWD types 2A, 2B, 2M followed at the Angelo Bianchi Bonomi Hemophilia Thrombosis Center of Milan, all characterized by RIPA and by specific mutations. Patients with VWD type 2M showed RIPA > 1.2 mg/ml, VWF:RCo/Ag < 0.7 VWFCB/Ag > 0.7; patients with VWD type 2B showed RIPA < 0.8 mg/ml, VWF:RCo/Ag < 0.7 VWFCB/Ag < 0.7; patients with VWD type 2A showed RIPA > 1.2 mg/ml, VWF:RCo/Ag < 0.7 VWFCB/Ag < 0.7. Collagen type I seems more sensitive than collagen type III in this setting of experiments. Therefore, differential diagnosis of VWD 2A, 2B, 2M can be obtained by using these tree tests together.
Conclusions. After discussion, there was agreement to publish the data of the WP on VWF assay in VWD diagnosis in a peer review Ms (future submission to JTH after presentation for formal approval by the ISTH-SSC on VWF in Geneva) and to go on with the WP on multimers and on collagen to standardize the minimal requirements and the appropriate collagen reagents for these two assays.
2) Proposal of WP on standardization on pre-analytical variables in VWF assays (G. Castaman)
Dr U. Budde and E. Favaloro presented data on the role of different temperature and processing-freezing-thawing of blood/plasma samples on VWF:RCo, VWF:CB and VWF multimers. After discussion there was a consensus that it is important to provide written recommendations about the best procedure to process blood and to prepare and store plasma samples to be devoted to VWF tests.
3) Final report of the WP on VWD Classification (David Lillicrap, Canada)
The final proposal for an updated VWD classification was presented by Evan Sadler and was formally approved by a unanimous vote Dr. Sadler will submit the Ms to JTH within two weeks.
4) Progress report of the WP on Molecular Biology and Expression Study (Anne Goodeve, UK)
Dr. L. Hilbert presented results of a survey; SSC/ISTH Working Party on VWF Molecular Biology and Expression Studies. The objective of this Working Party is to make a survey on the methods used for the identification and expression of von Willebrand disease (VWD) mutations. In 2005, 11 laboratories involved in 2 multicenter studies on type 1 VWD (European MCMDM-1VWD and Canadian projects) filled out a four page questionnaire to collect data on methodology. The major results from this pilot survey were presented during the SSC meeting in Sydney. A second questionnaire, with additional questions on both transient and stable transfection, was then sent not only to the participants from the pilot survey, but also to 21 other laboratories who had published data in this field. 22 laboratories from 15 countries responded and 19 laboratories completed the questionnaire. This international survey allows recommendations to be made on the conditions for the identification of a von Willebrand factor (VWF) mutation and the construction of a mutated expression vector. At present, no recommendations may be drawn for stable transfection because little data (4) is available. The 12 responses concerning transient transfection of VWF were heterogenous, so new participants must be recruited, and further discussion must take place before the best conditions are fixed. All interested parties, please contact hilbert@lfb.fr to participate. Dr. Hilbert will prepare a written report to be published as SSC Communication
Dr. S. Haberichter presented VWF pro-peptide assay for identification of type 1 VWD patients with decreased VWF survival. The decreased survival of VWF in plasma has recently been identified as a novel mechanism for type 1 VWD. We report four families with moderately severe type 1 VWD characterized by low plasma VWF:Ag and FVIII:C levels, proportionately low VWF:RCo, and dominant inheritance. A decreased survival of VWF in affected individuals was identified with VWF half-lives of 1-3 hours, while the half-life of VWF propeptide (VWFpp) was normal. DNA sequencing revealed a single (heterozygous) VWF mutation in affected individuals, S2179F in two families, and W1144G in two families. We report preliminary data establishing the normal range of VWFpp and VWFpp/VWF:Ag for each blood group. We demonstrate that the ratio of steady-state plasma VWFpp/VWF:Ag can be utilized to identify patients with a shortened VWF half-life. An increased ratio distinguished affected from unaffected individuals in all families. A significantly increased VWFpp/VWF:Ag ratio together with reduced VWF:Ag may indicate the presence of a true genetic defect and decreased VWF survival phenotype. This phenotype may require an altered clinical therapeutic approach and we propose to refer to this phenotype as type-1C VWD.
Proposal for the future work of this WP; Molecular screening recommendations in type 1 VWD Discussants: A Goodeve, D Lillicrap, P Collins
Dr. P. Collins presented the UK Haemophilia Centre Doctors’ Organization investigation into the molecular pathogenesis of type 1 VWD. Of 40 families diagnosed by UK Hemophilia Centers to have type 1 VWD 8 (20%) families were found not to have type 1 VWD following review. Six families were re-diagnosed to have type 2 VWD, 1 family was found to have a platelet storage pool disorder, and 1 family was determined to be unaffected. In the remaining 32 families direct DNA sequencing of the essential regions of the VWF gene identified a total of 11 different candidate mutations in index cases of 20 of the 32 confirmed type 1 VWD families. These included R1205H (4 families), Y1584C (8 families), R924Q (3 families), 2 other missense changes, 4 candidate splice site mutations and a single nt deletion. No candidate VWF gene mutation was identified in 12 of the 32 type 1 VWD index cases (38%). Linkage analysis showed that in 13 of the 32 families it was likely that VWD segregated with the VWF gene. In 8 families, VWD did not segregate with the VWF gene. Y1584C was present in 8 families but did not co-segregate with VWD in all cases. It was associated with blood group O in 95% of cases. In the rest of the cohort 70% of patients were blood group O.
Dr. A. Goodeve presented results of the MCMDM-1VWD study. 150 families with type 1 VWD were analyzed for mutations, and were identified in 105 (70%). A total of 124 candidate mutations were identified, of which 75 were different and of which 55 were novel. 18 (12%) index cases had 2 of more candidate mutations. 80% of the mutations were missense alterations, and these were located throughout VWF. Several changes were recurrent; R854Q (5 cases), R924Q (4 cases), C1130F/G/R (7 cases), R1205H (10 cases), R1374C/H (6 cases) and Y1584C (13 cases). Blood group O contributed significantly to low VWF, being present in 65% of index cases. In families lacking an identified mutation, blood group O was present in 78%, and in 95% of 19 families lacking a mutation and demonstrating non-linkage to VWF. Only a small proportion of families had candidate mutations demonstrating fully penetrant VWD.
Dr. D. Lillicrap presented a summary of the Type 1 VWD Canadian Cohort Study. One hundred and twenty three index cases and their families were studied and putative mutations identified within the VWF gene in 63% (n=78) of index cases leaving 37% (n=45) with no identified changes. These changes comprised 50 different putative mutations; 31 (62%) missense mutations, 8 (16%) changes involving the VWF transcriptional regulatory region, 5 (10%) small deletions/insertions, 5 (10%) splicing consensus sequence mutations, and 1 nonsense mutation. Twenty-one of the index cases had more than one putative VWF mutation identified. Although a total of 50 different mutations were identified in this study, twelve of the mutations occurred in multiple index cases. These twelve mutations were found in 63 (51%) of the index cases and, with one exception, these were missense mutations.
Presenters suggested that mutation screening of the VWF gene has limited general utility in genetic diagnostic and family studies in type 1 VWD. If genetic studies are performed, the incomplete penetrance and variable expressivity of type 1 VWD must be taken into account. Unless linkage of VWD phenotype with the VWF gene can be clearly demonstrated, the results of any genetic family studies should be interpreted with caution.
5) Progress report on Shear-stress Assays in VWD (Emmanuel Favaloro, Australia)
Dr. Z. M. Ruggeri discussed the general concepts for the use of shear-stress tests as a measurement of VWF function. He described the most recent data about the basic mechanisms on VWF-platelet interactions in different shear-rates conditions, from relatively low to high shear rates and about the role of VWF with different multimers on platelet adhesion (only dimeric VWF A1 domain) and platelet-platelet interactions (all the VWF multimers). He is positive about future applications of these methods in a more clinical setting to determine VWF defects in patients with VWD and suggest to make a joint WP with the SSC on biorheology.
Dr. A. Reininger in his presentation suggested that f uture diagnostic tools for the clinical application in VWD patients building on these new insights may therefore include various blood flow assays at elevated shear rates, and – although complicated – direct, real-time visualization and quantification.
Conclusion: Contacts with the SSC on Biorheology and vascular biology should be organized to make final statements about the application of these tests on VWD diagnosis
6) WP on ADAMTS-13 Assays ( Reinhard Schneppenheim , Germany)
Dr. R. Schneppenheim organized a very interesting session with presentation of the most recent work of several laboratories actively working on ADAMTS-13 assays.
The FRETS-VWF73 fluorescence assay was compared with a home made IRMA using full-length VWF for the measurement of ADAMTS13 activity in plasma samples from a cohort of 64 patients with thrombotic microangiopathies (TMA) including 41 acquired thrombotic thrombocytopenic purpura (TTP), 3 inherited TTP and 20 hemolytic uremic syndrome (HUS) and also from 20 normal subjects. Both methods were correlated (y = 0.94x + 1.71, correlation coefficient 0.97). With both methods, all normal subjects and most HUS patients showed ADAMTS13 activity higher than 50% although all TTP patients exhibited an ADAMTS13 activity lower than 5%. Compared to the IRMA method, the FRETS-VWF73 assay exhibits reliable results to measure low, moderately reduced as well as normal levels of ADAMTS13 activity. Accuracy and reproducibility of the assay combined with its short incubation time make the method highly attractive and appropriate for the clinical screening of patients with TMA.
Comparison of a slightly modified FRETS-VWF73 assay with older static assays using a full-length VWF substrate showed a good agreement between the ADAMTS-13 activity determined by FRETS-VWF73 and the older assays (Kremer Hovinga, Mottin and Lämmle, J Thromb Haemost. 2006;4:1146-8). However, the presence of hemoglobin in a patient’s sample constituted a problem in the FRETS-VWF73 assay and preliminary data indicate that discrepancies in ADAMTS-13 activity between different assays may be observed in rare cases of acute thrombotic microangiopathies.
The group developed a convenient and highly sensitive ELISA for ADAMTS13 activity. This ELISA precisely determined the half-life of infused plasma ADAMTS13 activity in USS patients to be between 2.3 and 3.5 days. A rapid fall in the level of ADAMTS13 activity after liver transplantation was found. Platelet transfusions in respective patients are therefore not recommended, but plasma infusions to supply ADAMTS13 after liver transplantation.
Plasma ADAMTS13 activities in the Japanese general population (N = 3,616) were measured by the FRETS-VWF73 assay. The values did not show a Gaussian distribution, suggesting that some kind of factor should affect the activity. The activities in males were lower than those in females. In both genders, the activities became lower with age. The plasma VWF level was not related to the ADAMTS13 activity. Comprehensive search revealed that Japanese people have six common missense polymorphisms. The P475S polymorphism significantly lowered plasma ADAMTS13 activity.
Zheng and his colleagues have shown that ADAMTS13 is synthesized and secreted from arterial, venous and microvascular endothelial cells; ADAMTS13 is secreted apically in polarized endothelial and epithelial cells; the apical secretion of ADAMTS13 depends on the CUB domains and their association with lipid rafts. Mutations in the CUB domains of ADAMTS13 may disrupt the signal directing polarized secretion, thereby leading to congenital ADAMTS13 deficiency and thrombotic thrombocytopenic purpura. Our data suggest that ADAMTS13 produced in the vascular endothelial cells may contribute significantly to the plasma levels of ADAMTS13 protease.
This study showed intracellular ADAMTS13 expression in podocytes in normal renal tissue and in the kidney of a TTP patient with the compound heterozygous mutation P353L and P457L. Expression of the mutants revealed impaired secretion and activity. Podocyte-derived ADAMTS13 may serve as a local protective mechanism in the high-shear microcirculation of the glomerulus. Deficient activity could result in the formation of platelet plugs in the glomeruli as seen in TTP.
The enhanced proteolysis of VWF in patients with classic VWD type IIA is due to an intrinsic susceptibility of mutant VWF to ADAMTS13. VWF type 2B is proteolyzed to a similar extent, however the underlying mechanism has not been experimentally elucidated. The results of a study on ADAMTS13 proteolysis of recombinant VWF 2B and VWF IIE, the latter being a phenotype with decreased proteolysis suggest that in a static assay both mutants are proteolyzed roughly normal. Therefore, the different phenotypes are most probably due to different function under flow conditions: enhanced proteolysis of VWF type 2B as a result of enhanced affinity to platelets and decreased proteolysis of VWF IIE due to its poor functional activity in platelet dependent hemostasis, respectively.
Physiological concentrations of NaCl inhibit the hydrolysis of VWF by ADAMTS-13. It was demonstrated that chloride ions specifically bind to the A1 domain of VWF. Cl- ions stabilize a “super-folded” conformation of VWF through binding to the A1 domain. The affinity of Cl- for the VWD R1306 mutant was reduced and its hydrolysis by ADAMTS-13 accelerated. These findings showed that Cl- ions control allosterically the availability of the Y1605-M1606 bond to the proteolytic attack of ADAMTS-13.
After a fruitful discussion among the participants and the audience, Dr. Schneppenheim proposed issues of ADAMTS13 and VWF of current interest:
7) Progress report on WP on Acquired von Willebrand syndrome (J. Di Paola, USA) Dr U. Budde presented the technical problems on the measurements of auto-antibodies in AVWS: he showed that the use of plasma-derived or recombinant VWF used to capture auto-antibodies from the patients serum or plasma can be critical for the assay due to the role of blood groups. He proposes to use recombinant VWF from insects. Dr. C Mazurier proposes to use plasma-derived VWF from donors with blood group O.
Dr. K. Mertens, on behalf of Dr. J van Mourik , reported on the significance of the measurement of the plasma concentration of the pro-peptide of VWF (VWAgII) in the diagnosis of acquired von Willebrand syndrome (AVWS). An increased steady state pro-peptide/VWF ratio is clearly indicative of decreased survival of mature VWF, as encountered in AVWS or certain congenital VWF defects, and useful as an index to assist in the diagnosis of abnormal VWF catabolism. The molar concentration of plasma pro-peptide was assessed by calibrating pooled normal plasma against recombinant pro-peptide purified to homogeneity. This approach allows straightforward standardization of pro-peptide assays.
Dr R.R. Montgomery reported his experience with his assay with VWF pro-peptide that was tested together with native VWF:Ag in normal individuals with different blood groups. This assay will be commercially available soon. After a general discussion, a standardization of these assays was proposed for the use in AVWS and also in patients with different forms of inherited VWD.
8) Progress reports and new proposal of WP and clinical surveys (T. Abshire, USA)
Dr. G. Castaman presented the progress report of the WP on Biological response versus clinical efficacy of DDAVP in VWD type 1 and 2; 128 patients have been enrolled. The dead line for enrollment is postponed to 31 st December 2006. The study is recruiting patients through the web site – www.ddavp.in.vwd.com.
Dr, A.B. Federiciproposed a new WP on the clinical & molecular markers of VWD type 3 with frequency of alloantibodies to be organized as a joint project of ISTH-SSC and WFH
Dr. T. Abshire proposed a new WP on the use of prophylaxis in VWD. This project is composed by a retrospective survey and by a prospective study on the use of different VWF concentrates in the secondary long-term prophylaxis of hemarthrosis, epistaxis and G I bleeding in severe VWD patients with a VWF:RCo < 20 U/dL.
9) Other reports and proposals, concluding remarks (A. B. Federici, Italy)
Dr. A. B. Federici presented the concluding remarks of the entire two sessions, summarizing all studies and proposals to be reported next year in Geneva.