Alessandra Casonato, Department of Medical and Surgical Sciences, Via Ospedale Civile 105, Padua, Italy. E-mail: firstname.lastname@example.org
Reduced von Willebrand factor (VWF) half-life has been suggested as a new pathogenic mechanism in von Willebrand disease (VWD). The usefulness of VWF propeptide (VWFpp) in exploring VWF half-life was assessed in 22 type 1 and 14 type Vicenza VWD patients, and in 30 normal subjects, by comparing the findings on post-Desmopressin (DDAVP) VWF t1/2 elimination (t1/2el). The VWFpp/VWF antigen ratio (VWFpp ratio) was dramatically increased in type Vicenza VWD (13·02 ± 0·49) when compared to normal subjects (1·45 ± 0·06), whereas it appeared to be normal in all type 1 VWD patients (1·56 ± 0·7), except for the four carrying the C1130F mutation (4·69 ± 0·67). A very short VWF t1/2el was found in type Vicenza VWD (1·3 ± 0·2 h), while all type 1 VWD patients had a t1/2el similar to that of the controls (11·6 ± 1·4 and 15·4 ± 2·5 h respectively), except for the four patients carrying the C1130F mutation, who had a significantly shorter VWF survival (4·1 ± 0·2 h). A significant inverse correlation emerged between VWFpp ratio and VWF t1/2el in both VWD patients and normal subjects. The VWFpp ratio thus seemed very useful for distinguishing between type 1 VWD cases with a normal and a reduced VWF survival, as well as for identifying type Vicenza VWD.
Von Willebrand factor (VWF) is a large, adhesive, multimeric glycoprotein that takes part in the haemostatic process by promoting platelet adhesion at sites of vascular injury. It also serves as a carrier of factor VIII (FVIII) in the circulation. VWF is synthesised and stored in endothelial cells and megakaryocytes (Bloom et al, 1973; Nachman et al, 1977). It is synthesised as pre-pro-VWF with a 22-amino-acid signal peptide, a 741-amino-acid propeptide, and a 2050-amino-acid mature VWF protein (Wagner, 1990; Voorberg et al, 1991). Pro-VWF is subject to extensive post-translational modifications, including signal peptide cleavage, C-terminal dimerisation, carbohydrate processing, sulphation and amino-terminal multimerisation (Wagner et al, 1986; Voorberg et al, 1990). Due to proteolytic processing in the acid compartment of the trans-Golgi network, pro-VWF is further separated into two polypeptides, the VWF propeptide (VWFpp) and mature VWF. VWFpp is non-covalently associated with mature VWF multimers and both proteins are stored in the α-granules of platelets, or in the Weibel-Palade bodies of endothelial cells, from where VWF is released via either a constitutive or a regulated pathway (Wagner & Marder, 1984; Visher & Wagner, 1994). At the cell level, the main function of the propeptide is to facilitate the amino-terminal multimerisation of mature VWF within the post-Golgi compartments and its targeting to Weibel-Palade storage bodies in endothelial cells (Hannah et al, 2002). After secretion into the plasma, VWFpp dissociates from VWF and circulates as a homodimer at concentrations around 1 μg/ml with a half-life of 2–3 h, whereas mature VWF circulates at about 10 μg/ml with a half-life of 8–14 h (Borchiellini et al, 1996; Haberichter et al, 2006).
Reduced levels or functional abnormalities of VWF are associated with a bleeding tendency known as von Willebrand disease (VWD), the most common inherited bleeding disorder with an estimated prevalence as high as 1% in the general population. VWD is multi-faceted and is currently classified according to the structural and/or functional anomalies of the mature VWF (Sadler, 2006). VWD types 1 and 3 are quantitative defects: type 1 is characterised by a partial quantitative VWF deficiency, usually coinciding with an autosomal dominant inheritance, a variable penetrance and a highly variable phenotype; type 3 is characterised by a complete lack of VWF. Type 2 VWD refers instead to a qualitative VWF deficiency and is divided into types 2A, 2B, 2M and 2N (Sadler, 2006), though this distinction is not always clear, and both qualitative and quantitative defects occur in some cases (Casonato et al, 2003).
More recently, Haberichter et al (2006) claimed that a shorter VWF survival can be predicted from the ratio of VWFpp to VWF concentrations in the plasma.
The present studies were conducted to determine the VWFpp ratio and its correlation with VWF survival in patients with type 1 and type Vicenza VWD.
Materials and methods
Patients and healthy volunteers were enrolled to the study after obtaining their written informed consent in accordance with the Helsinki Declaration and after the study had been approved by our institutional review board.
Blood was drawn from the antecubital vein and anticoagulated using 3·8% sodium citrate (1:10, vol/vol). Platelet-poor plasma (PPP) was obtained by centrifugation at 800 g for 15 min, then at 12 000 g for 4 min to eliminate cell fragments, and stored at −80°C for subsequent measurements.
Plasma and platelet VWF antigen (VWF:Ag) were measured with a home-made enzyme-linked immunosorbent assay (ELISA) method (Casonato et al, 1992), using a horseradish peroxidase (HRP)-conjugated anti-VWF antibody (Dako, The Netherlands). VWF ristocetin cofactor activity (VWF:RCo) was measured in normal washed and formalin-fixed platelets and ristocetin at 1·0 mg/ml using an aggregometric method. Factor VIII (FVIII) coagulant was measured using a one-stage method, with cephaloplastin as activated cephalin. VWF collagen binding capacity (VWF:CB) was assessed by ELISA using type I and type III collagen diluted in acetic acid (95% and 5% solutions respectively). Briefly, after overnight coating with collagen, microtitre plates were incubated with plasma VWF for 1 h at room temperature; bound VWF was evaluated with HRP-conjugated anti-VWF antibody (Dako).
The concentration of VWFpp in plasma was determined using an ELISA (a kind gift from GTI Diagnostics, Waukesha, WI, USA). Briefly, prediluted calibrators and diluted plasma samples were added to microwells coated with monoclonal antibodies specific for VWFpp; bound VWFpp was assessed with biotinylated anti-VWFpp monoclonal antibody and streptavidin-labelled HRP. Reportable results are given in U/dl, taking the first reference curve dilution as 100 U/dl.
Desmopressin (DDAVP; Emosint, Sclavo, Italy) was administered subcutaneously at a dose of 0·3 μg/kg to characterise VWF half-life. Blood samples were collected before and at 15, 30, 60, 120, 180, 240, 360 and 480 min, and 24 h after administering the DDAVP (Michiels et al, 2002). The time courses of plasma VWF concentrations were analysed using a one-compartment model with first-order input and output kinetics (Gibaldi & Perrier, 1975), in which baseline concentrations, B, were also incorporated as follows: plasma concentration = A x (e−Kelx t−Krex t) + B, where A is the y-axis intercept, Kre is the release rate constant, Kel is the elimination rate constant, and t is the time. The model was fitted to each set of concentration-time data using the Prism statistical package (GraphPad, San Diego, CA, USA). Goodness of fit was evaluated by r2. Elimination half-life (t1/2el) was calculated using the standard formula: t1/2el = 0·693/Kel.
Human genomic DNA was isolated from peripheral blood leucocytes using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). All 52 exons of VWF, including the intron–exon boundaries and the 3′ and 5′ regulation sites, were amplified using primers chosen according to the VWF sequence determined by Mancuso et al (1989). Amplification products were sequenced in both directions using a BigDye Terminator sequencing kit (Applied Biosystem, ABI, Warrington, UK) and ABI 3130 xl Genetic Analyser. VWF cDNA nucleotides were numbered, taking as +1 the A of the ATG initiation codon located 250 nucleotides downstream from the transcription cap site, as recommended by the International Society on Thrombosis and Haemostasis (ISTH) VWF Scientific and Standardization Committee (Goodeve et al, 2001).
Laboratory data and pharmacokinetic parameters were expressed as mean ± standard error (SE). Student’s t-test and correlations were performed, as appropriate. Welch’s correction was used when variances were not equal. P-values below 0·05 were considered statistically significant.
Thirty-six patients with VWD (22 type 1 and 14 type Vicenza) and 30 normal individuals were studied; their main haemostatic findings are given in Table I. Type 1 VWD was diagnosed according to the following criteria: a decrease in plasma VWF:Ag, VWF:CB and VWF:RCo levels, with normal VWF:RCo to VWF:Ag and VWF:CB to VWF:Ag ratios, and a life-long history of bleeding. Type Vicenza VWD patients were identified by: a marked reduction in plasma VWF:Ag, VWF:RCo and VWF:CB levels, normal platelet VWF content and larger than normal VWF multimers. Type Vicenza cases were confirmed by genetic analysis enabling the identification of both M740I (G2220A) and R1205H (G3614A) mutations in exons 17 and 27 of VWF.
Table I. Main haemostatic findings observed in VWD patients and normal subjects investigated.
In type 1 VWD patients, who belonged to 12 unrelated families and were almost all in blood group O (20/22), low VWF:Ag, VWF:CB and VWF:RCo levels were observed, together with a reduced platelet VWF content (48·0 ± 9·1 U/dl, vs. normal range 70–140 U/dl) (Table I). VWF analysis revealed mutations throughout VWF; six patients had nonsense mutations, 11 had missense mutations and one had compound missense and nonsense mutations (Tables I and II); no mutations were detectable in four patients, however, in either VWF or the 3′ and 5′ regulation sites (Table II).
Table II. VWF mutations and corresponding VWFpp ratios and VWF half-lives in VWD patients.
Type of mutation
VWF:Ag t1/2el (h)
1534-3 C>A; del Ex 14, frameshift to terminus
1·1 ± 0·1
14·1 ± 2·1
2763 C>A; C 921 X
1534-3 C>A; del Ex 14, frameshift to terminus
7130 ins C, frameshift to terminus
Stop codon/missense mutation
1534-3 C>A; del Ex 14, frameshift to terminus
7085 G>T; C 2362F
3389 G>T; C 1130F
4·7 ± 0·7
4·1 ± 0·2
3433 C>T; R 1145 C
6187 C>T; P 2063 S
1·7 ± 0·1
12·1 ± 0·3
7085 G>T; C 2362 F
1·8 ± 1·3
8·3 ± 2·4
8113 G>A; G 2705 R
1·8 ± 0·1
6·7 ± 1·4
2220 G>A; M 740 I
13·0 ± 0·5
1·3 ± 0·2
3614 G>T; R 1205 H
The 14 type Vicenza VWD patients came from eight unrelated families and all belonged to non-O blood groups. They all showed ultralarge VWF multimers (data not shown), and their main haemostatic findings are given in Table I.
In the normal controls, the mean VWF:Ag levels were lower in those belonging to the O blood group than in those belonging to non-O blood groups (P < 0·01) and the same was true of VWF:CB (P < 0·05), while no significant differences emerged for FVIII (Table I).
VWF half-life in healthy controls and VWD patients
DDAVP was administered and time courses of VWF:Ag, VWF:CB and FVIII levels were recorded for up to 24 h. The administration of DDAVP prompted a significant increase in VWF and FVIII levels in both patients and normal controls but, after reaching a peak, VWF showed different elimination times in the different study groups, so t1/2el was used as the main pharmacokinetic parameter for exploring VWF half-life (Fig 1). In healthy controls, VWF t1/2el ranged from 5·3 to 31·5 h (mean 15·4 ± 2·5 h), and O group individuals had a lower t1/2el than non-O cases (9·4 ± 1·0 h vs. 21·4 ± 4·4 h), (P < 0·0001). In type 1 VWD patients, the mean VWF half-life was statistically no different from that of the normal subjects (Fig 2). Within the type 1 VWD group, four patients had a significantly shorter VWF half-life than the others (t1/2el 4·1 ± 0·2 h, P < 0·005): these four patients belonged to three unrelated families and, at the heterozygous level, carried the G>T 3389 mutation in exon 26, which substitutes a cysteine with a phenylalanine at position 1130 of VWF (C1130F) (Table II). Their mean VWF:Ag, VWF:CB and FVIII values (28·0 ± 9·8, 14·2 ± 5·2 and 52·7 ± 23·4 U/dl respectively) were lower, though not to a statistically significant degree, than those of the other type 1 VWD patients (40·3 ± 5·7, 39·9 ± 5·6 and 62·0 ± 7·9 U/dl respectively). An even more pronounced drop in VWF survival emerged in the type Vicenza VWD patients, whose mean t1/2el was significantly lower than in controls (1·3 ± 0·2 h, P < 0·0001) (Table II).
VWFpp and VWFpp/VWF:Ag ratio
The ratio of VWFpp to VWF:Ag (or VWFpp ratio) can be used to predict a shorter than normal VWF half-life, so we investigated the VWFpp ratio in our VWD patients and normal subjects, also seeking a correlation with VWF half-life (Table III). In healthy subjects, the VWFpp levels ranged from 62 to 197 U/dl (mean 102·7 ± 5·1 U/dl), with a mean VWFpp ratio of 1·45 ± 0·06 (Fig 3). After grouping subjects by ABO blood group, there was no significant difference in the mean VWFpp levels between O and non-O cases, but the VWFpp ratio differed significantly between O (1·6 ± 0·06) and non-O individuals (1·26 ± 0·04) (P < 0·005) (Table III).
Table III. Mean VWF, VWFpp, VWFpp ratio and VWF half-lifes in the VWD patients and normal subjects.
Type 1 (n = 18)
Type 1-C1130F (n = 4)
Type Vicenza (n = 14)
All normals (n = 30)
O Group (n = 17)
Non-O Group (n = 13)
40·3 ± 5·5
27·6 ± 10·0
12·1 ± 1·9
96·3 ± 6·9
79·3 ± 8·6
113·2 ± 9·4
55·4 ± 7·1
105 ± 20·3
136·5 ± 21·9
102·7 ± 5·1
99·8 ± 7·9
106·6 ± 5·8
1·56 ± 0·07
4·69 ± 0·67
13·02 ± 0·49
1·45 ± 0·06
1·6 ± 0·06
1·26 ± 0·04
VWF t1/2el (h)
11·6 ± 1·4
4·1 ± 0·2
1·3 ± 0·2
15·4 ± 2·5
9·4 ± 1·0
21·4 ± 4·4
In type 1 VWD, patients with a normal VWF survival had significantly lower VWFpp levels than controls or patients with a shorter VWF half-life (55·4 ± 7·1, 102·7 ± 5·1 and 105 ± 20·3 U/dl respectively). On the other hand, the VWFpp ratio differed in different type 1 VWD patients, i.e. those carrying the C1130F mutation had a significantly higher VWFpp ratio (4·69 ± 0·67) than either type 1 VWD patients with a normal VWF survival (1·56 ± 0·07) or controls (1·45 ± 0·06) (P < 0·001) (Table III). The situation was much the same when type 1 VWD patients (who were mainly blood group O) were compared with controls belonging to the O blood group. It is worth adding that genetic characterisation of three of the four C1130F VWD patients was prompted by the identification of the first unrelated patient with a similarly increased VWFpp ratio and reduced VWF survival.
A dramatic increase in VWFpp ratio was apparent in the type Vicenza VWD cases: the VWFpp ratio ranged from 7·14 to 17·7, mean 13·02 ± 0·49 – 10 times higher than in the control group (P < 0·001) (Fig 3), whereas their mean VWFpp levels (136·5 ± 21·9 U/dl, with values ranging between 71 and 193 U/dl) were not dissimilar from those of controls and patients with type 1 VWD and a reduced VWF survival.
A significant inverse correlation (P < 0·0001) emerged between the VWFpp ratio and the VWF:Ag values in VWD patients and healthy subjects (Fig 4 upper panel); the VWFpp ratio and VWF t1/2el also correlated inversely in normal individuals and VWD patients (P < 0·0001), the highest VWFpp ratio coinciding with the lowest VWF half-life (Fig 4 lower panel). Such a finding suggests that the VWFpp ratio changes in much the same way, whatever the VWF half-life.
The results of this study demonstrated that the steady-state ratio of plasma VWFpp to VWF can be used to identify VWD patients characterised by a reduced VWF survival. This applies not only to type Vicenza VWD, which has a very short VWF half-life and a very high VWFpp ratio, but also for patients with a mildly reduced VWF half-life. Since a higher VWFpp ratio correlates with a shorter VWF half-life, it becomes an excellent tool not only for investigating VWF survival, but also for characterising VWD.
It has recently been suggested that a shorter VWF survival may be one of the mechanisms responsible for VWD (Casonato et al, 2002; Lenting et al, 2004). The impact of a shorter VWF half-life in determining the VWD phenotype was first demonstrated clearly in type Vicenza VWD, a variant characterised by a considerably reduced VWF survival but normal VWF synthesis (Casonato et al, 2002). A shorter VWF survival has also been reported in type 1 VWD (Brown et al, 2003), as well as in other forms of VWD associated with VWF mutations, such as C1130F, C1149R and C2671Y (Van Schooten et al, 2005; Haberichter et al, 2006), though the latter mutations also coincide with a decline in VWF synthesis or an impaired intracellular VWF transportation.
A proper VWF survival analysis takes at least 24 h to enable the main pharmacokinetic parameters to be calculated reliably, as the observation time must be no shorter than the normal half-life of VWF (Borchiellini et al, 1996; Gallinaro et al, 2008). This was the approach used in the present study, but it is troublesome and time-consuming, and not always feasible (Gibaldi & Perrier, 1975; Casonato et al, 2002). The VWFpp ratio has recently been proposed as a means for evaluating VWF half-life, since a higher ratio can predict a shorter VWF survival in patients with type 1 VWD (Haberichter et al, 2006, 2008). The steady-state levels of plasma VWF and VWFpp are, in fact, the outcome of the secretion and clearance of both molecules; given the different half-life of VWF and its propeptide, any variation in VWF or both molecules is likely to affect said ratio. Our results confirm the validity of this hypothesis and also demonstrate that VWF survival and the VWFpp ratio correlate well. Type Vicenza VWD patients (who have a very short VWF half-life) had a very high VWFpp ratio – around 10 times the ratio found in normal subjects. In type 1 VWD, the picture was more complex: most type 1 VWD patients had a VWFpp ratio similar to that of controls, but a few (carrying the C1130F mutation) had a higher VWFpp ratio coinciding with a shorter VWF survival, though this was less pronounced than in the type Vicenza cases. Type 1 VWD probably defines a very heterogeneous group, including both pure VWF reductions (with no change in the structure and function of the residual VWF), as in gene null mutations (Gallinaro et al, 2006), and also forms of VWD characterised by an abnormal VWF molecule, such as those associated with missense mutations (Haberichter et al, 2006; Casonato et al, 2007). All the type 1 VWD patients we studied had lower platelet VWF levels, suggesting a decrease in VWF synthesis, but a few of them (those carrying the C1130F mutation) also had a shorter VWF half-life. None of the type 1 VWD patients carrying VWF null mutations (stop codon) had an abnormal VWF half-life; on the other hand, not all the missense VWF mutations studied were associated with a shorter VWF survival. This latter observation suggests that whether a missense VWF mutation affects VWF survival cannot be predicted from the nature of the defect or its consequences in terms of VWF multimer organisation, as shown by patients with an altered multimer structure, like those carrying VWF C2362F mutations (Casonato et al, 2007). Instead, the mutation location appears to be crucial, considering that most of the mutations reported to date as being associated with a shorter VWF survival occur in the D3 domain (R1205H, C1130F, C1149R, W1144G) (Haberichter et al, 2006, 2008; Castaman et al, 2008).
The VWFpp ratio thus enables patients with a shorter VWF survival to be identified among type 1 VWD cases, but the ratio appears to be particularly useful in characterising VWD in cases with the type Vicenza variant. Type Vicenza VWD is currently identified on the basis of the discrepancy between platelet and plasma VWF levels, and the presence of ultralarge VWF multimers, even when no VWF half-life assessments or genetic investigations are available (Casonato et al, 2006). Given its marked increase, the VWFpp ratio could be used as a specific marker to easily identify type Vicenza VWD.
A significant correlation emerged between the extent to which the VWFpp ratio increased and the corresponding reduction in VWF survival – patients with the shortest VWF half-life (e.g. type Vicenza cases) had the highest VWFpp ratio, whereas patients with intermediate VWFpp ratios had a smaller reduction in VWF survival. The correlation was maintained in healthy subjects indicating that the VWFpp ratio may be a general predictor of VWF survival, independently of the mechanism(s) responsible for variable elimination rate (Gallinaro et al, 2008).
Being able to document a shorter VWF survival has major implications in VWD, in terms of grading its severity and treatment decisions, as patients with a combination of a reduced VWF synthesis and a shorter VWF survival are likely to have a more severe form of the disease. Measuring how long it takes for DDAVP to take effect by assessing VWF half-life also enables us to predict how long this treatment can assure a satisfactory haemostasis, so the availability of an easy tool for investigating VWF survival may be of great help in the management of VWD patients.
In conclusion, the VWFpp ratio appears to be very useful for investigating VWF survival and identifying VWD patients with a shorter VWF half-life. The extent to which the VWFpp ratio increases (as seen in type Vicenza and in type 1 VWD associated with C1130F mutations) also helps to characterize the nature of the VWF defect.
This work was supported by grants from the Telethon Foundation (Rome, Italy).