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von Willebrand factor collagen binding assay in von Willebrand disease type 2A, 2B, and 2M

2006; Elsevier BV; Volume: 4; Issue: 9 Linguagem: Inglês

10.1111/j.1538-7836.2006.02069.x

1538-7933

Luciano Baronciani, Augusto B. Federici, Giovanna Cozzi, Maria Teresa Canciani, P.M. Mannucci,

Heparin-Induced Thrombocytopenia and Thrombosis

Commenting on our description of a novel type 2B mutant of the von Willebrand factor (VWF) gene (R1308L) characterized by defective collagen binding (VWF:CB) [1Baronciani L. Federici A.B. Beretta M. Cozzi G. Canciani M.T. Mannucci P.M. Expression studies on a novel type 2B variant of the von Willebrand factor gene (R1308L) Characterized by defective collagen binding.J Thromb Haemost. 2005; 3: 2689-94Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar], Favaloro [2Favaloro E.J. 2B or not 2B? What is the role of VWF in platelet‐matrix interactions? And what is the role of the VWF:CB in VWD diagnostics? These are the questions.J Thromb Haemost. 2006; 4: 892-3Abstract Full Text Full Text PDF PubMed Google Scholar] raises two items: the role of VWF in platelet–collagen interactions and the option of classifying this novel variant on the basis of VWF:CB plasma levels [2Favaloro E.J. 2B or not 2B? What is the role of VWF in platelet‐matrix interactions? And what is the role of the VWF:CB in VWD diagnostics? These are the questions.J Thromb Haemost. 2006; 4: 892-3Abstract Full Text Full Text PDF PubMed Google Scholar]. In order to comment on these items, we have recently re‐investigated our patients with von Willebrand disease (VWD) carrying mutations within the part of the VWF gene encoding the A1 domain and diagnosed as VWD type 2B or 2M on the basis of enhanced (< 0.8 mg mL−1 ristocetin) or reduced (> 1.2 mg mL−1 ristocetin, normal range being 0.8–1.2) ristocetin‐induced platelet agglutination (RIPA) in platelet‐rich plasma (PRP) [3Federici A.B. Castaman G. Mannucci P.M. Italian Association of Hemophilia Guidelines for the diagnosis and management of von Willebrand disease in Italy.Haemophilia. 2002; 8: 607-21Crossref PubMed Google Scholar, 4Castaman G. Federici A.B. Rodeghiero F. Mannucci P.M. Willebrand's disease in the year 2003: towards the complete identification of gene defects for correct diagnosis and treatment.Haematologica. 2003; 88: 94-108PubMed Google Scholar]. For comparison, we have also investigated a group of patients with type 2A VWD, carrying mutations within the VWF A2 domain. To measure the plasma levels of ristocetin cofactor (VWF:RCo) we used an in‐house method based upon formalin‐fixed platelet agglutination [5Macfarlane D.E. Stibbe J. Kirby E. Zucker M.B. Grant R.A. McPherson J. A method for assaying von Willebrand factor (ristocetin cofactor).Thromb Diath Haemorrh. 1975; 34: 306-18Crossref PubMed Google Scholar] or, when plasma VWF:Ag levels were < 10 U dL−1, the more sensitive VWF:RCo ELISA [6Federici A.B. Canciani M.T. Forza I. Mannucci P.M. Marchese P. Ware J. Ruggeri Z.M. A sensitive ristocetin co‐factor activity assay with recombinant glycoprotein Ibalpha for diagnosis of patients with low von Willebrand factor levels.Haematologica. 2004; 89: 77-85PubMed Google Scholar]. To assay plasma VWF:CB we have used two in‐house methods, one with 95% type I and 5% type III equine collagen (Nycomed 7806141; Nyomed Italy srl, Milan, Italy), (indicated as VWF:CB‐I) [6Federici A.B. Canciani M.T. Forza I. Mannucci P.M. Marchese P. Ware J. Ruggeri Z.M. A sensitive ristocetin co‐factor activity assay with recombinant glycoprotein Ibalpha for diagnosis of patients with low von Willebrand factor levels.Haematologica. 2004; 89: 77-85PubMed Google Scholar], and the other with human collagen type III (Southern Biotechnologies – RB123061; Southern Biotechnologies Associates, Birmingham, AL, USA) (indicated herewith as VWF:CB‐III). All these values and those of VWF antigen (VWF:Ag) measured by ELISA [6Federici A.B. Canciani M.T. Forza I. Mannucci P.M. Marchese P. Ware J. Ruggeri Z.M. A sensitive ristocetin co‐factor activity assay with recombinant glycoprotein Ibalpha for diagnosis of patients with low von Willebrand factor levels.Haematologica. 2004; 89: 77-85PubMed Google Scholar] were calculated against the Fifth VWF International Standard (NIBSC). The results, expressed as mean values ± SD of RIPA (mg mL−1) and of ratios of VWF:RCo, VWF:CB‐I and VWF:CB‐III to VWF:Ag, are summarized in Table 1.Table 1Single or mean values (±SD) of RIPA and VWF ristocetin cofactor and collagen binding assays normalized to antigen values (ratios) in relation to VWF gene mutations in VWD type 2A, 2B, and 2MMutationsRIPA (mg mL−1)VWF:RCo/VWF:AgVWF:CB‐I/VWF:AgVWF:CB‐III/VWF:AgType 2M (n = 20)L1278P (1)2.000.251.000.83R1315C (1)1.900.450.900.85D1277E‐L1278del (2)1.80 ± 0.200.13 ± 0.030.74 ± 0.010.78 ± 0.02Y1321C (2)1.40 ± 0.100.16 ± 0.011.12 ± 0.390.98 ± 0.19R1315L (4)1.60 ± 0.200.20 ± 0.100.70 ± 0.270.71 ± 0.07R1374H (10)1.80 ± 0.200.20 ± 0.100.71 ± 0.180.83 ± 0.12Type 2B (n = 28)R1341Q (1)0.700.650.290.48R1308C (4)0.70 ± 0.100.50 ± 0.100.10 ± 0.040.35 ± 0.14V1316M (4)0.50 ± 0.100.40 ± 0.300.10 ± 0.040.34 ± 0.22R1306W (11)0.60 ± 0.100.50 ± 0.100.12 ± 0.100.74 ± 0.24P1337L (1)*0.700.830.520.70R1341W (1)*0.700.490.390.99R1308L (3)*0.60 ± 0.200.80 ± 0.200.56 ± 0.030.87 ± 0.37I1309V (3)*0.40 ± 0.100.60 ± 0.200.47 ± 0.170.87 ± 0.09Type 2A (n = 14)I1628T (1)3.000.050.090.31G1629R (1)1.300.060.110.21R1597W (2)1.90 ± 0.800.30 ± 0.020.17 ± 0.010.52 ± 0.21V1607D (3)1.30 ± 0.100.25 ± 0.150.14 ± 0.170.42 ± 0.19V1665E (3)2.50 ± 0.800.17 ± 0.110.09 ± 0.100.41 ± 0.13S1506L (4)2.30 ± 0.300.25 ± 0.170.05 ± 0.010.37 ± 0.14Normal values0.80–1.20> 0.70> 0.70> 0.70Values between parentheses indicate the number of patients with that mutation; patients with a relatively normal presence of high molecular weight VWF multimers.Ag, antigen; CB, collagen binding; RCo, ristocetin cofactor; RIPA, ristocetin‐induced platelet agglutination; VWD, von Willebrand disease; VWF, von Willebrand factor. Open table in a new tab Values between parentheses indicate the number of patients with that mutation; patients with a relatively normal presence of high molecular weight VWF multimers. Ag, antigen; CB, collagen binding; RCo, ristocetin cofactor; RIPA, ristocetin‐induced platelet agglutination; VWD, von Willebrand disease; VWF, von Willebrand factor. In VWD type 2M (n = 20), diagnosed on the basis of decreased RIPA and a full set of VWF multimers [3Federici A.B. Castaman G. Mannucci P.M. Italian Association of Hemophilia Guidelines for the diagnosis and management of von Willebrand disease in Italy.Haemophilia. 2002; 8: 607-21Crossref PubMed Google Scholar, 4Castaman G. Federici A.B. Rodeghiero F. Mannucci P.M. Willebrand's disease in the year 2003: towards the complete identification of gene defects for correct diagnosis and treatment.Haematologica. 2003; 88: 94-108PubMed Google Scholar], plasma VWF:RCo was always lower than VWF:Ag (VWF:RCo/Ag ratios < 0.7), whereas VWF:CB‐I and VWF:CB‐III were similar to VWF:Ag (VWF:CB/Ag ratios ≥ 0.7), whatever the type of collagen. These data indicate that in VWD type 2M (or at least in our cases with mutations within VWF A1 domain), the VWF:RCo assay is the most discriminant in order to reveal the presence of a dysfunctional VWF, while VWF:CB‐I and VWF:CB‐III are not, because their results roughly coincided with those of VWF:Ag. So far, only one family of two members with type 2M VWD and low plasma VWF:CB in the presence of borderline (30–60 U dL−1) VWF:RCo was reported [7Ribba A.‐.S. Loisel I. Lavergne J.‐.M. Juhan‐Vague L. Obert B. Cherel G. Meyer D. Girma J.‐.P. Ser968Thr Mutation within A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen.Thromb Haemost. 2001; 86: 848-54Crossref PubMed Google Scholar]. These abnormalities were associated with a mutation in the VWF A3 domain, and low VWF:CB was also found in the expressed recombinant mutant VWF S1731T [7Ribba A.‐.S. Loisel I. Lavergne J.‐.M. Juhan‐Vague L. Obert B. Cherel G. Meyer D. Girma J.‐.P. Ser968Thr Mutation within A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen.Thromb Haemost. 2001; 86: 848-54Crossref PubMed Google Scholar]. Therefore, isolated VWF:CB defects appear to be uncommon. In VWD type 2B (n = 28), diagnosed on the basis of enhanced RIPA in PRP [3Federici A.B. Castaman G. Mannucci P.M. Italian Association of Hemophilia Guidelines for the diagnosis and management of von Willebrand disease in Italy.Haemophilia. 2002; 8: 607-21Crossref PubMed Google Scholar, 4Castaman G. Federici A.B. Rodeghiero F. Mannucci P.M. Willebrand's disease in the year 2003: towards the complete identification of gene defects for correct diagnosis and treatment.Haematologica. 2003; 88: 94-108PubMed Google Scholar], the VWF:RCo/Ag ratio was below normal (< 0.7) in several but not all patients, whereas VWF:CB‐I/Ag ratios were lower than VWF:RCo/Ag ratios in all patients, whether multimers were intact or not. The VWF:CB‐III assay was less sensitive than the VWF:CB‐I assay (VWF:CB‐III/Ag ratios higher than VWF:CB‐I/Ag ratios) in sorting out these defects of collagen binding in VWD 2B, particularly so in patients with a relatively normal set of multimers (Table 1). In VWD type 2A (n = 14), diagnosed on the basis of reduced RIPA and lack of large and intermediate molecular weight multimers [3Federici A.B. Castaman G. Mannucci P.M. Italian Association of Hemophilia Guidelines for the diagnosis and management of von Willebrand disease in Italy.Haemophilia. 2002; 8: 607-21Crossref PubMed Google Scholar, 4Castaman G. Federici A.B. Rodeghiero F. Mannucci P.M. Willebrand's disease in the year 2003: towards the complete identification of gene defects for correct diagnosis and treatment.Haematologica. 2003; 88: 94-108PubMed Google Scholar], both VWF:RCo and VWF:CB‐I were very low, yielding very low ratios to VWF:Ag (Table 1). Also, in these patients VWF:CB‐III was less sensitive than VWF:CB‐I in the identification of dysfunctional VWF, yielding higher ratios to VWF:Ag. The VWF:RCo/Ag ratios of patients with type 2A were clearly lower than those with 2B VWD, whereas the VWF:CB‐I/Ag ratios were similar, except for those 2B patients who had a full set of VWF multimers (Table 1). This pattern of results was observed in spite of the fact that in type 2A VWD the multimeric structure of plasma VWF was more markedly defective than in type 2B VWD. With this background information on the VWF:CB values obtained in our type 2M, 2B, and 2A patients with mutations identified within A1 and A2 domains (Table 1), we can now attempt to tackle two questions: (i) how often are collagen binding defects present in type 2B VWD?; and (ii) which is the role of VWF:CB assays in VWD diagnosis? Our data indicate that VWF:CB‐I defects are found not only in the 'unusual' type 2B variant R1308L, previously described by us [1Baronciani L. Federici A.B. Beretta M. Cozzi G. Canciani M.T. Mannucci P.M. Expression studies on a novel type 2B variant of the von Willebrand factor gene (R1308L) Characterized by defective collagen binding.J Thromb Haemost. 2005; 3: 2689-94Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar], but also in the majority of type 2B patients (Table 1). We surmize that type 2B mutations in the A1 domain might also affect, through the adjacent A2 domain, the conformation of the A3 domain, resulting in an impaired binding to collagen. On the other hand, VWF:CB does not appear to be defective in our type 2M patients, at least when type I and III collagens are used. As the VWF A1 domain also contains the binding site for collagen type VI [8Mazzuccato M. Spessotto P. Masotti A. De Apollonia L. Cozzi M.R. Yosshioka A. Perris R. Colombatti A. De Marco L. Identification of domains responsible for von Willebrand Factor Type VI collagen interaction mediating platelet adhesion under high flow.J Biol Chem. 1999; 274: 3033-41Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar], it would be of interest to test these patients with this type of collagen. The nature of such a markedly different behavior of the 2M and 2B mutations, sometimes located next to each other within the VWF A1 domain (R1315L is associated with type 2M whereas V1316M is associated with type 2B), remains to be understood. Finally, we would like to make a few comments on the role of the plasma VWF:CB assay in the diagnosis of VWD, and on its possible use in screening patients suspected of having a defective primary hemostasis. In our laboratory VWF:CB, introduced for diagnostic purposes in addition to VWF:RCo since 1998, has proved to be a useful additional test to distinguish not only type 2A from 2M but also type 2A from 2B [9Federici A.B. Canciani M.T. Forza I. Cozzi G. Ristocetin cofactor and collagen binding activities normalized to antigen levels for a rapid diagnosis of type 2 von Willebrand disease: single center comparison of four different assays.Thromb Haemost. 2000; 84: 1127-8Crossref PubMed Scopus (0) Google Scholar]. In the last few years, we have measured VWF:CB in 40 patients with unexplained prolonged bleeding time (with normal platelet function assays) and found no plasma VWF:CB defect. An additional important issue related to the use of VWF:CB in the diagnosis of VWD patients is that results vary according to the type of collagen sources, as reported by Favaloro [10Favaloro E.J. Collagen binding assay for von Willebrand factor (VWF:CB): detection of von Willebrand disease (VWD) and discrimination of VWD subtypes, depends on collagen sources.Thromb Haemost. 2000; 83: 127-35Crossref PubMed Scopus (0) Google Scholar] and demonstrated also by the results of this study. Hence, there is little evidence at the moment that VWF:CB should be used instead of VWF:RCo in the general screening of patients suspected of carrying a defect of primary hemostasis. The authors state that they have no conflict of interest.