Measurement of von Willebrand factor binding to a
recombinant fragment of glycoprotein Iba in an enzyme-linked
immunosorbent assay-based method: performances in patients
with type 2B von Willebrand disease
von Willebrand factor (VWF) is a large multimeric glycopro- tein present in plasma, a-granules of platelets, Weibel–Palade bodies in endothelial cells and subendothelial matrices. It plays a major role in the process of platelet adhesion by interacting with both the subendothelium and the glycoprotein (GP) Ib receptor on platelets. In vivo, plasma VWF does not bind to platelet receptors, but its initial binding to collagen and/or other subendothelial matrix components in areas of high- blood shear stress, appears to be the trigger for the binding to GPIb. This VWF/GPIb interaction can be studied in vitro by using ristocetin as an agonist (Scott et al, 1991). The domain of VWF that interacts with the GPIb receptor is present on a trypsic fragment corresponding to amino acid residues V1212–
K1491 (Fujimura et al, 1986). The cysteine loop (C1272– C1458) in the A1 domain of VWF, has been identified as an important element in the regulation of binding to GPIb. The region of GPIb involved in this interaction has been identified to the amino-terminal 42 kDa globular part of GPIba (Vicente et al, 1990).
Defects in VWF result in von Willebrand disease (VWD), the most common inherited bleeding disease. The current classification (Sadler, 1994) distinguishes quantitative VWF defects, partial in VWD type 1 or complete in type 3, and qualitative ones in type 2, with four major subtypes (2A, 2B, 2M and 2N). Type 2N refers to variants with a markedly decreased affinity of VWF for factor VIII. Type 2A VWD refers Claudine Caron,1Lysiane Hilbert,2Karen
Vanhoorelbeke,3Hans Deckmyn,3Jenny Goudemand1and Claudine Mazurier2
1Department of Haematology, University Hospital,2Laboratoire Franc¸ais du Fractionnement et des Biotechnologies, Lille, France, and3Laboratory for Thrombosis Research, KU Leuven Campus Kortrijk, Kortrijk, Belgium
Received 19 December 2005; accepted for publication 23 February 2006
Correspondence: Claudine Caron, Laboratoire d’He´matologie, Hoˆpital Cardiologique, Bd du Professeur J. Leclercq, 59037 Lille Cedex, France. E-mail: [email protected]
Summary
Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIba) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIba. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0Æ2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1Æ8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.
Keywords: von Willebrand factor, von Willebrand disease, enzyme-linked immunosorbent assay, glycoprotein Ib, ristocetin.
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to variants with decreased platelet-dependent function that is associated with the absence of high-molecular weight (HMW) VWF multimers, whereas type 2M refers to variants with decreased platelet-dependent function that is not caused by the absence of the HMW VWF multimers. In contrast, type 2B refers to variants with increased affinity of VWF for platelet GPIb generally resulting in spontaneous binding of VWF to platelets that form aggregates, detected as a loss of plasma HMW VWF multimers and mild thrombocytopenia. In vitro, this increased affinity allows a ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP) at lower concentrations of ristocetin than needed for normal PRP (Ruggeri et al, 1980).
The classification of a type 2 VWD (except subtype 2N) is first suggested by a reduced ristocetin co-factor activity of VWF (VWF:RCo) compared with the VWF antigen (VWF:Ag) level, with a ratio of VWF:RCo to VWF:Ag usually lower than 0Æ7, according to the guidelines recently proposed for Italy by Federici et al (2002). The measurement of VWF:Ag is easy to perform by well standardised and sensitive enzyme-linked immuosorbent assay (ELISA) meth- ods using poly/monoclonal anti-VWF antibodies for which automated methods on a latex agglutination basis have also been developed, allowing rapid determination in urgent cases (Veyradier et al, 1999). The VWF:RCo classically is per- formed on normal platelets mixed with patient platelet poor plasma (PPP), but this is a time-consuming assay, based on the measurement of platelet agglutination in an aggregometer (MacFarlane et al, 1975) or by a rapid macroscopical slide technique (Brinkhous et al, 1975) using preferentially fixed platelets rather than fresh ones. Furthermore, this test is hampered by an unacceptable poor reproducibility. Recently, we described a more sensitive, reliable and reproducible ELISA method (Vanhoorelbeke et al, 2000) that uses a recombinant GPIba fragment immobilised on a microtitre plate. To further classify a patient in a specific VWD type 2 subtype, more advanced investigations are required, such as the VWF collagen-binding (VWF:CB) assay, the plasma VWF multimeric analysis, and the RIPA assay. The RIPA assay enables the identification of patients that display an enhanced aggregation response at low ristocetin concentration. This paradoxal response is the main feature of type 2B VWD but also of platelet-type pseudo-VWD, where a gain-of-function of the GPIba is present (Weiss et al, 1982). To differentiate type 2B VWD from pseudo-VWD, a direct measurement of the capacity of the patient plasma VWF to support ristocetin- induced aggregation of normal washed platelets, has been proposed (Weiss & Sussman, 1986). An indirect method measures, by ELISA, residual-free VWF present in the supernatants (Ruggeri et al, 1980). Furthermore, Meulien et al (1992) described a ristocetin-dependent platelet-binding assay in which recombinant VWF, preincubated with a radiolabelled monoclonal antibody (MAb) against VWF, is added to formalin-fixed platelets in the presence of increasing concentrations of ristocetin. In this method, VWF associated
radioactivity bound to platelets is measured in the pellet. These methods have been widely used, with some minor modifications, to study biochemical characteristics of plasma VWF–GPIb interaction (Mohri et al, 1989; Matsushita & Sadler, 1995; Miura et al, 2000; Nakayama et al, 2002), or to test expressed recombinant VWF with various candidate mutations (Hilbert et al, 1995, 1998, 2002; Hillery et al, 1998; Facey et al, 1999; Ribba et al, 2001; Stepanian et al, 2003). However, these methods have some limitations for diagnostic laboratories, such as the need of patient’s fresh platelets for RIPA or preparation of washed normal platelets, and licence for radioactivity handling.
We present here a new assay as an alternative for the study of the ristocetin-induced VWF binding to platelets. In the ELISA assay set up to measure VWF:RCo (Vanhoorelbeke et al, 2000) fresh or formaldehyde-fixed normal platelets are no longer required as the binding of VWF to a recombinant fragment of GPIb (His1–Val289) (rfGPIba), purified as described by Schumpp (Schumpp-Vonach et al, 1995) is measured. This fragment also binds to VWF in the presence of botrocetin, with a comparable affinity as the proteolytic 42 kDa fragment of the purified human platelet GPIb/V/IX complex (Schumpp-Vonach et al, 1995). Furthermore, in contrast to the previously described platelet-based assay (Meulien et al, 1992), here rabbit anti-human VWF antibodies labelled with an enzyme are used rather than radioactivity. This test is able to readily distinguish type 1 from type 2 VWD samples (Vanhoorelbeke et al, 2002). However, so far, further differentiation of type 2 subtypes was not possible.
We report on the results obtained with this new test in 31 type 2B VWD patients with eight known mutations located in or close to the A1 disulphide loop, and we show that, this test can be used to discriminate between normal plasma and plasma of type 2B VWD patients.
Materials and methods
Normal and VWD patients plasma samples
Blood samples were collected by venepuncture in tubes containing 3Æ8% (0Æ129 mol/l) sodium citrate; PPP was obtained after double centrifugation at 2000 g for 15 min, and was stored at )80C until use. Normal plasmas were obtained from 12 voluntary donors (EFS-Nord de France, Lille, France) with different ABO blood groups, to prepare the normal plasma pool (NPP). Plasma samples from 31 type 2B VWD patients from 15 families were studied. All patients displayed a positive RIPA at 0Æ5 mg/ml ristocetin concentra- tion. Eight different mutations (H1268D, R1306W, R1306Q, R1306L, R1308C, V1316M, R1341Q and A1461V) in the exon 28 of VWF gene have been previously identified in these families (Meyer et al, 2001) (Table I). As a control of non- enhanced binding, seven type 2A VWD patients with three different mutations (R1315C, R1597W and L1639D) were also tested.
Platelet lysates
Platelets were prepared from 15% ethylene diaminetetraacetic acid blood samples. PRP was obtained after centrifugation at 200 g for 15 min and the platelet number was counted. The PRP was then centrifuged at 3000 g for 10 min and the platelet pellet was stored at )80C. Before use, the pellet was resuspended and lysed in phosphate-buffered saline (PBS) containing 0Æ5% Triton X-100 to adjust the platelet concen- tration to 3 · 109/ml and centrifuged at 10 000 g for 5 min. The VWF:Ag level in the supernatant was measured by ELISA (Mazurier et al, 1980). Platelet lysates from six normal subjects
and 13 type 2B VWD patients from families 4, 7, 9, 10, 11, 12, 13 and 15 were studied (Table I).
Ristocetin-induced binding of VWF to rfGPIba assay A polystyrene plate (Maxisorp Nunc, Rochester, NY, USA) was coated, overnight at 4C, with 100 ll of 5 lg anti-GPIba MAb 2D4 in 50 mmol/l carbonate buffer pH 9Æ6. MAb 2D4 has a conformation-dependent epitope and has no inhibitory activ- ity on ristocetin-induced human platelet agglutination (Van- hoorelbeke et al, 2000). The plate was then saturated with 3% Table I. Biological description of the 2B VWD patients.
Mutation in pre-pro VWF
Family member
Age (years)
Platelets number (109/l)
Ratio VWF:RCo /VWF:Ag
% HMW multimers*
OD ratio
(a) Plasma VWF (b) Platelet VWF 0 mg/ml 0Æ2 mg/ml 0 mg/ml 0Æ2 mg/ml
H1268D 1-1 75 274 0Æ29 1Æ09 3Æ24
R1306W 2-1 47 150 0Æ45 2Æ01 4Æ61
2 13 202 0Æ49 1Æ22 2Æ95
3-1 50 0Æ57 15 0Æ82 1Æ47
2 26 0Æ66 15 1Æ18 2Æ09
R1306Q 4-1 19 242 0Æ70 70 1Æ69 4Æ17 5Æ22 4Æ08
2 40 266 0Æ67 48 1Æ04 3Æ06 2Æ51 4Æ64
3 34 188 0Æ83 72 1Æ46 3Æ60 3Æ14 5Æ51
4 11 180 0Æ54 50 1Æ62 2Æ13
R1306L 5-1 36 178 0Æ58 44 1Æ47 3Æ20
R1308C 6-1 11 160 0Æ28 8 2Æ14 2Æ88
7-1 37 180 0Æ46 1Æ04 1Æ12 2Æ69 2Æ94
8-1 2 467 0Æ18 8 1Æ66 2Æ72
V1316M 9-1 32 100 0Æ20 10 2Æ61 3Æ82 4Æ93 4Æ32
2 1 0Æ30 31 1Æ97 3Æ39
R1341Q 10-1 17 125 0Æ59 43 1Æ50 4Æ35 1Æ95 5Æ26
2 22 153 0Æ74 35 2Æ67 4 1Æ34 3Æ92
3 44 235 0Æ67 26 1Æ65 4Æ02 2Æ93 3Æ1
11-1 55 188 0Æ50 27 1 1Æ87 0Æ74 2Æ06
2 6 394 0Æ42 34 1Æ41 2Æ43
3 2 324 0Æ32 17 1Æ18 2Æ28
4 33 286 0Æ36 22 1Æ47 2
5 62 187 0Æ49 22 1Æ32 1Æ95
12-1 42 213 0Æ41 32 1Æ30 1Æ37 2Æ30 2Æ65
2 17 160 0Æ73 31 1Æ08 2Æ67
13-1 21 274 0Æ43 22 1Æ63 2Æ34 2Æ80 3Æ38
14-1 40 71 0Æ56 1Æ15 6Æ85
2 43 80 0Æ62 1Æ33 3Æ63
3 18 214 0Æ44 2Æ22 2Æ21
A1461V 15-1 66 47 0Æ76 63 4Æ76 12Æ25 2Æ10 2Æ6
2 74 177 0Æ72 10 2Æ28 6Æ91
All n 31 28 31 24 31 31 12 12
Mean 32 204 0Æ51 31Æ4 1Æ64 3Æ40 2Æ72 3Æ71
SD 21 92 0Æ17 18Æ8 0Æ75 2Æ13 1Æ29 1Æ10
VWD, von Willebrand disease; VWF, von Willebrand factor; RCo, ristocetin co-factor; HMW, high-molecular weight; Ag, antigen.
*Percentages relative to a pool of normal plasmas.
Ratio of OD of the patient sample when compared with OD of the NPP (a) or normal platelet lysate (b) inserted in the same assay.
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milk powder (in 50 mmol/l PBS pH 7Æ4–250 ll/well) for 90 min at room temperature (RT), followed by a 2 h incubation at 37C with a solution of 1 lg/ml of rfGPIba in PBS, 0Æ1% Tween-20 (TBS) (100 ll/well). Meanwhile, 500 ll of either NPP or the test PPP sample – adjusted to 7 IU/dl VWF:Ag (in TBS) – were mixed with 10 ll of a 0Æ2 mg/ml dilution of anti-VWF-HRP (horse radish peroxidase-conju- gated rabbit anti-human VWF polyclonal antibodies; Dako, Glostrup, Denmark). The non-inhibitory activity of the anti- VWF-HRP on the VWF binding has been preliminarily verified (data not shown). After a 1-h incubation at RT, 50 ll of the mixture and 50 ll of a serial ristocetin (Diagnos- tica Stago, Asnie`res, France) dilution (0–1 mg/ml at final concentration) were added to the wells coated with rfGPIba for an incubation of 20 min at 37C. After each incubation step, the plate was washed five times with TBS. The detection of captured ristocetin-VWF–anti-VWF HRP complexes on the rfGPIba was done with ortho-phenylenediamine (3 mg/ml in 40 mmol/l sodium citrate – 10 mmol/l citric acid pH 5 and 0Æ03% H2O2 – 100 ll/well) and the colouring reaction was stopped with H2SO4(2 mol/l). The absorbance (OD) was read at 490 nm. The results of the VWF binding were expressed in OD as a function of ristocetin concentration. For evaluation of the type 2B VWD phenotype, the ratio of OD of the patient plasma sample over the OD of the NPP measured in the same assay (ratio OD sample/OD NPP) was calculated at the low ristocetin concentration of 0Æ2 mg/ml.
Results
Ristocetin-induced binding of normal samples (plasma and platelets)
The binding of VWF from normal plasmas (n¼ 12) and from NPP (13 different experiments performed in a 18-month period) to the captured rfGPIba in the presence of increasing concentrations of ristocetin was evaluated. Figure 1 shows the mean and SD of the results obtained. As expected, no binding
of plasma VWF to rfGPIba was observed in the absence of agonist and this binding was ristocetin-dose dependent.
When platelet lysates containing platelet VWF, from six normal individuals were tested, there was still no binding in the absence of ristocetin but the dose-dependent ristocetin- induced binding gave OD values that were higher than those obtained with NPP, as shown in Fig 1.
Binding of type 2B plasma or platelet VWF to rfGPIba Patients with the R1341Q mutation
The ability of plasma VWF from type 2B VWD patients to bind to rfGPIba in the presence of increasing ristocetin concentrations was, for all the 14 patients (families 10 to 14) with the R1341Q mutation (Fig 2) the most informative; the graphics have been amplified in the area between 0 and 0Æ5 mg/ml of ristocetin. Binding of R1341Q-VWF from each patient was compared with that obtained with the NPP tested in the same experiment. Typical curves were obtained with significant enhanced binding at the lowest ristocetin concen- trations with the most discriminant response at 0Æ2 mg/ml ristocetin. So, the ratio (OD sample/OD NPP) in the absence of ristocetin (for evaluation of spontaneous binding) or at 0Æ2 mg/ml ristocetin (for evaluation of 2B VWD phenotype) was calculated. Ratios for all samples tested, and the indicated mutation are given in Table I. In contrast to NPP or normal platelets, spontaneous binding to rfGPIba may occur with patients sample, irrespective of the mutation and was without significant difference between the mutations, with a mean OD ratio of 1Æ64 (SD¼ 0Æ75) in 2B plasmas and 2Æ72 (SD ¼ 1Æ29) in 2B platelets.
These 14 patients have been tested for the capacity of plasma VWF to bind to rfGPIba (Fig 2A–D). In families 10 (Fig 2A) and 11 (Fig 2B), all relatives displayed increased binding at 0Æ2 mg/ml ristocetin; the OD ratio varied between 4 and 4Æ35 for family 10 and between 1Æ87 and 2Æ43 for family 11. In families 12 (Fig 2C) and 14 (Fig 2D), some variability in plasma VWF affinity was observed between the relatives,
Fig 1. Ristocetin-induced binding of von Willebrand factor in normal plasma and platelet lysates (mean and SD). ( ) Normal individual plasmas, ( ) normal plasma pool and (r) normal platelet lysate.
although, they shared the same molecular abnormality. Indeed, patient 12-1 shows only a slight increase at low ristocetin concentrations (ratio at 0Æ2 mg/ml¼ 1Æ37) whereas this ratio was 2Æ67 for patient 12-2. Patient 14-1 displayed a greater affinity for rfGPIba (ratio at 0Æ2 mg/ml¼ 6Æ85) than his relative 14-3 (ratio only of 2Æ21). Patient 13-1 (Fig 2C), the only subject tested in this family, showed a typical binding curve with an OD ratio of 2Æ34 at 0Æ2 mg/ml ristocetin. On the whole, except for patient 12-1, the OD ratio at 0Æ2 mg/ml ristocetin was >1Æ8 for all the patients belonging to families 10 to 14 (Table I). Thus, we considered that a ratio above 1Æ8 might be indicative for a type 2B phenotype.
Platelet lysate samples. Six patients (from families 10 to 13) have been tested for the ability of their platelet VWF to bind to rfGPIba. The increased binding at low ristocetin concentrations, typical of type 2B VWD, was clearly evident in all the platelet lysate samples tested (ratio at 0Æ2 mg/ml >2 – 2Æ06 to 5Æ26). It is noteworthy that, platelet lysate from patient 12-1, with a plasma VWF ratio of 1Æ37 at 0Æ2 mg/ml, displayed a significant increased platelet VWF binding at 0Æ2 mg/ml ristocetin, with a ratio of 2Æ65 (Fig 3).
Patients with mutations at position R1306
Three different mutations at position 1306 have been identified in the patients included in this study. Patients from families 2 and 3 harboured the R1306W mutation, whereas patients from families 4 and 5 displayed the R1306Q and R1306L mutations, respectively, (Table I). An enhanced binding of plasma VWF at 0Æ2 mg/ml ristocetin (ratio >2 – 2Æ09 to 4Æ61) was observed for all patients except for patient 3-1, with a ratio of 1Æ47.
Platelet lysate samples. Only platelets from three of the four relatives in family 4 were available for study. VWF in all platelet lysates showed an increased affinity with ratios of >4 (4Æ08–5Æ51) at 0Æ2 mg/ml ristocetin.
Patients with other mutations The H1268D, R1308C, V1316M and A1461V mutations were reported in six families included in this study (Table I).
Plasma samples. Whereas, patient 7-1 with the R1308C mutation did not show an increased ratio at 0Æ2 mg/ml ristocetin (ratio¼ 1Æ12), the two other R1308C patients displayed values >2Æ7. For the two patients with the V1316M Fig 2. Binding of plasma von Willebrand factor from type 2 von Willebrand disease patients with the R1341Q mutation. Family 10 in (A) member 10-1 (r); 10-2 ( ); 10-3 (d) with respective intra-assay performed normal plasma pool (NPP) ( ; D; ). Family 11 in (B) member 11-1(r); 11-2 ( ); 11-3 (d); 11-4 (+); 11-5 ( ) with respective intra-assay NPP ( ; h; ; +; D). Families 12 and 13 in (C) member 12-1(r); 12-2( ); 13-1(d) with respective intra-assay NPP ( ; (; s). Family 14 in (D): member 14-1(r); 14-2( ); 14-3(d) with respective intra-assay NPP ( ; h; ).
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mutation, this value was >3 whereas for the patients with the A1461V mutation, it was 12Æ25 for 15-1 and 6Æ91 for 15-2 (Table I). It is noteworthy that, patient 15-1 also displayed the highest spontaneous binding (ratio in the absence of ristocetin¼ 4Æ76).
Platelet lysate samples. Platelet VWF from patients 7-1 (Fig 3), 9-1 and 15-1 exhibited the typical increase (ratio >2Æ5) at 0Æ2 mg/ml ristocetin.
Binding of type 2A plasma or platelet VWF to rfGPIba As expected, all the seven type 2A VWF plasmas tested did not show an increased OD ratio at 0Æ2 mg/ml ristocetin; further- more, this ratio was always lower than 1 (from 0Æ68 to 0Æ98) and similar to that obtained in normal plasmas (from 0Æ71 to 1Æ02).
Negative correlation between the increased affinity of patient VWF for rfGPIba and the platelet count
Platelet count varied from 47 · 109/l to 467 · 109/l (mean ± SD: 213 ± 92Æ109/l) in the 28 type 2B VWD patients studied (Table I); only five patients (patients 9-1, 10-1, 14-1, 14-2 and 15-1) had thrombocytopenia (<150 · 109/l). The plasma VWF from these five patients displayed high binding at 0Æ2 mg/ml ristocetin, with a ratio ranging from 3Æ63 to 12Æ25. VWF from patient 15-1, with the most severe thrombo- cytopenia (47 · 109/l) showed the highest binding to rfGPIba (ratio at 0Æ2 mg/ml ristocetin¼ 12Æ25). It is noteworthy that the three patients aged £10 years (patients 8-1, 11-2 and 11-3), had the highest platelet counts; 467, 394 and 324 · 109/l respectively. The ratio at 0Æ2 mg/ml of plasma VWF was very similar in these three patients (between 2Æ28 and 2Æ72).
Furthermore, there was a significant negative correlation (r¼)0Æ596; P ¼ 0Æ016) between the platelet count and the OD ratio at 0Æ2 mg/ml ristocetin, in type 2B VWD patients
>10 years in age.
Absence of correlation between the increased affinity of patient VWF for rfGPIba and the percentage of HMW multimers
The percentage of HMW multimers above 10 mers was evaluated in samples from 24 patients (Table I). No correlation between the extent of the lack in HMW multimers and the enhanced affinity of VWF for rfGPIba at 0Æ2 mg/ml ristocetin could be found (r¼ 0Æ353, P ¼ 0Æ077).
Discussion
Type 2B VWD refers to qualitative variants with increased affinity of VWF for platelet GPIb. These patients are generally characterised by an increased RIPA as well as an enhanced binding capacity of plasma VWF to platelets at low ristocetin concentrations. The RIPA uses PRP and has to be performed as soon as possible (within 2 h) after the sample is taken. Furthermore, RIPA does not discriminate between a plasma VWF hyperaffinity for GPIb (type 2B VWD) and a platelet GPIb hyperaffinity for VWF (pseudo-VWD). Therefore, evaluation of the ability of the plasma VWF to bind to platelets is usually performed to discriminate between these two abnormalities. The previously reported methods require radiolabelled anti-VWF antibodies and normal formaldehyde-fixed platelets (Meulien et al, 1992; Miura et al, 2000; Hilbert et al, 2002; Stepanian et al, 2003), which limits their straightforward applicability in most laboratories because of the need for a radioactivity licence and access to normal platelets for in vitro use.
Fig 3. Binding of von Willebrand factor from platelet lysates from type 2 von Willebrand disease patients. Members 7-1 ( ) and 11-1 ( ) with intra- assay normal platelet lysate (D); member 12-1(r) with intra-assay normal platelet lysate ( ).
We describe here a new method to measure ristocetin- induced binding of plasma or platelet VWF to GPIb that overcomes these limitations. We have previously shown (Vanhoorelbeke et al, 2000) that the VWF:RCo measurement can be performed using a recombinant fragment of GPIb that has been captured by an anti-GPIb antibody, and revelation of the VWF–GPIb complex by commercial HRP-labelled anti- VWF antibodies. We have also demonstrated (Vanhoorelbeke et al, 2002) that, with this test in combination with the determination of VWF antigen levels, one can readily distin- guish type 1 from type 2 VWD. The present study has confirmed that, despite some modifications, normal plasma or platelet VWF binds to rfGPIba in the presence of ristocetin and that this interaction is dose-dependent. This modified test can be used to identify patients with type 2B VWD mutations, where the ratio OD patient/OD NPP, determined at 0Æ2 mg/ml ristocetin and at a constant concentration of 0Æ70 IU/dl VWF:Ag, was found to be a very good indicator to identify type 2B VWD patients. Indeed, plasma VWF from 28/31 type 2B VWD patients with known mutations showed an enhanced OD ratio (>1Æ8) at 0Æ2 mg/ml ristocetin.
In plasma of type 2B VWD patients, however, the larger, more active VWF multimers are no longer present and this may confound the observations. Nevertheless, as the normal range of VWF multimers is present in the platelet granules (De Groot et al, 1989), the platelet VWF-binding capacity may be more straightforward. Indeed, VWF in the platelet
lysates from all twelve patients tested showed an enhanced binding to rfGPIba, including two of the three patients that were not identified when using their plasma. For the last patient, no such evidence could be obtained despite the fact that the results of all the other members of the family were unequivocal after re-evaluation. Our results further confirm that some variability in this ratio level may exist not only between patients with different mutations, but also between patients with the same mutation and even in patients from the same family. Such variability has been already reported with previous tests (Gaucher et al, 1995).
On the other hand, it was also noted that the two patients with the highest responses (ratio¼ 6Æ91 and 12Æ25), had only subnormal VWF:RCo/VWF:Ag ratios, which did not initially suggest a type 2 VWD, and had the A1461V mutation, which is located outside the A1 loop (Hilbert et al, 1995).
As it is known that type 2B VWD induces a loss of HMW multimers and mild thrombocytopenia, we evaluated whether a correlation exists between the OD ratio at 0Æ2 mg/ml and these two features. Indeed, plasma VWF reactivity for rfGP1ba in the presence of low doses of ristocetin may be relative to the importance of the lack in HMW multimers. So, in VWF with A1461V mutation there is only a mild decrease in HMW multimers as observed in patient 15-1. Consequently, the VWF still reacts strongly with platelets and rfGP1ba in the presence of low doses of ristocetin. So, although we did not find
Fig 4. Flow chart to identify the various types and subtypes of von Willebrand disease (VWD). Undetectable plasma level of von Willebrand factor antigen (VWF:Ag) identifies type 3 VWD. If VWF:Ag is present, a proportionate level of reduced ristocetin co-factor activity of VWF (VWF:RCo), with also a proportionate level of VIII:C, diagnoses type 1 VWD; if VIII:C is lower than VWF:Ag, a type 2N VWD has to be investigated. When VWF:RCo is lower than VWF:Ag, a type 2 VWD is evocated; further tests enable the various subtypes (2M, 2A and 2B) to be determined. VWF collagen binding (VWF:CB) and VWF multimers pattern are normal only in type 2M; ristocetin-induced platelet aggregation (RIPA) and VWF binding to glycoprotein (GP) Ib at low-ristocetin concentration are positive only in type 2B; type 2A is characterised by decreased VWF:CB level, abnormal VWF multimers pattern and negative RIPA or VWF binding to GPIb at low ristocetin concentration.
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statistical correlation between the percentage of residual HMW multimers and the OD ratio at 0Æ2 mg/ml ristocetin, it is possible that the interpretation of our test may be less easy in patients harbouring more reactive point mutations, as the major lack of the HMW multimers does not enable a reaction with GPIb. In these cases, it will be useful to test the VWF from patient platelets. Effectively, platelet VWF from type 2B patients, similar to endothelial VWF, is normally multimerised and has an increased affinity for platelet GP1b, even in the absence of ristocetin (De Groot et al, 1989).
It has been previously documented in a large kindred (Mazurier et al, 1988) that platelet count is age-dependent, with the occurrence of thrombocytopenia only in adult affected members. This was confirmed in the present study by the absence of thrombocytopenia (platelet count
>300 · 109/l) in three patients younger than 10 years. On the other hand, a relationship was found between OD at 0Æ2 mg/ml ristocetin and thrombocytopenia (platelet count
<150 · 109/l) in patients older than 10 years from five families, where the more reactive type 2B plasmas were linked to more severe thrombocytopenia.
In conclusion, this study has extended the applicability of the test to enable the diagnosis of type 2B VWD by further optimisation. This test has several advantages over the classical methods as: (i) it can be performed on frozen plasma samples, without the need to obtain fresh PRP as required for a RIPA test; (ii) it is simple to perform as it does not use radiolabelled anti-VWF antibodies, and is therefore, accessible for laboratories without a licence to use radioactive material and (iii) it is sensitive, as 28/31 known type 2B patients showed typical plasma-binding curves with an enhanced OD ratio (>1Æ8) at 0Æ2 mg/ml ristocetin. Furthermore, it may be also applied in biochemical VWF interactions studies on rVWF expressed to reproduce various candidate VWD mutations. Figure 4 summarises the tests that need to be successively performed for the accurate diagnosis of types and subtypes of VWD.
Acknowledgements
We are very grateful to Drs A.Voyer and J.Die´val (Amiens), ME Briquel (Nancy), C. Boinot (Poitiers), K. Pouymayou (Marseille), L. Rugeri (Lyon) for providing VWD patient plasmas, and to S. Hermoire and D. Grenier for excellent technical assistance.
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