Von Willebrand disease type Vicenza: In search of a classification for the archetype of reduced von Willebrand factor survival

Abstract Type Vicenza von Willebrand disease (VWD) features a von Willebrand factor (VWF) with a very short half‐life, and is classified as a form of type 1 VWD. To test the appropriateness of type Vicenza VWD classification, the main features of 17 patients from eight unrelated families were analysed. They had low VWF antigen levels and function (always below 20 U/dl); ristocetin‐induced platelet aggregation sometimes normal, sometimes reduced/absent (even in the same patient); normal platelet VWF levels; an increased VWF propeptide to VWF antigen ratio (8.74 ± 1.65 vs. normal 1.04 ± 0.28) and a reduced VWF half‐life. Plasma VWF multimer levels were homogeneously reduced, and unusually large VWF multimers were sometimes present. Recombinant p.R1205H VWF showed a normal synthesis, release, function, and multimer pattern, with no ultra‐large VWF multimers. The mathematical model by Galvanin et al. was used to explore the kinetic changes in VWF after DDAVP. It showed that the release, but especially the proteolysis (k proteol 1.0−3 ± 2.5−3 vs. normal 4.5−4 ± 6.4−4) and elimination (k el 1.0−2 ± 5.2−3 vs. normal 1.1−3 ± 6.8−4) of type Vicenza VWF were significantly higher than normal. The increased elimination is consistent with the short half‐life, while the increased proteolysis was unexpected. As a shorter survival of VWF is wholly responsible for the type Vicenza VWD phenotype (VWF synthesis, structure and function are normal), it might be better to classify it as a type 2 VWD (rather than type 1) to emphasise the greater interaction with clearance receptors as a new VWF functional defect.

the highest haemostatic capacity [2]. A defective VWF identifies von Willebrand disease (VWD), the most common inherited bleeding disorder. VWD can be characterised by quantitative VWF defects (type 1 and type 3) or VWF functional abnormalities (type 2) [3].
Type 1 VWD is defined as a partial quantitative VWF defect, with the residual VWF featuring no functional abnormalities, and a normal or near-normal multimer pattern [4][5][6].
When type Vicenza VWD was first described by Mannucci et al. in 1985 [7], it was defined as a 'platelet-normal type 1 VWD' , underscoring that the normal platelet VWF content coincided with a significant reduction in circulating VWF. The large VWF multimers were all present, sometimes with the presence of unusually large VWF oligomers as well [8]. It was later reclassified as type 2M VWD because of the abnormal ristocetin-induced platelet aggregation (RIPA) seen in these patients. The defect was found associated with the p.R1205H mutation [9,10], usually combined with the p.M740I mutation [11], which is now considered a polymorphism. It was ultimately demonstrated that type Vicenza VWD is characterised by a very short VWF survival-the main reason for the low circulating VWF levels [12]. An accelerated VWF clearance seems to be the only explanation for the presence of ultra-large multimers in type Vicenza VWD [13]. It is now classified once again as a type 1 VWD, based on low circulating VWF levels and the presence of all VWF multimers [3].
Ever since type Vicenza VWD was first described, its classification has been evolving, and a final solution probably has yet to be found [14].
In the present contribution, we analyse all the known features of VWF in cases of type Vicenza VWD to establish the appropriateness of its current classification.

MATERIALS AND METHODS
Patients and normal subjects were studied in accordance with the Helsinki Declaration, after obtaining their written informed consent.

Haemostatic analysis
The main haemostatic findings in the patients considered here have been reported elsewhere [15]. Plasma and platelet VWF antigen The results are given in U/dl, taking the first reference curve dilution as 100 U/dl [18]. VWF multimers were analysed by electrophoresis on 1.6% high-gelling temperature agarose containing 0.1% sodium dodecyl sulphate. The multimers were detected by autoradiography after reaction with anti-VWF polyclonal antibody (DAKO) labelled with 125 I, and viewed with the DS-50000 Epson densitometer scanner.

Genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes using

Expression experiments
The pSVvWFA plasmid containing normal human full-length VWF cDNA was mutated by recombinant PCR, as previously described [20]. For the expression studies, the pSVH1205 VWF was tran-

Mathematical model
The time course of post-DDAVP VWF:Ag and VWF:CB was analysed with a two-compartment, physiologically based model derived from the one proposed by Galvanin et al. [21], which can characterise the release, proteolysis and clearance mechanisms of VWF, and its multimer distribution (see Supporting Materials). This model comprises a system of differential and algebraic equations, and each subject is characterised by three main pharmacokinetic (PK) constants: the VWF release rate constant k rel , the proteolysis rate constant k proteol , and the elimination rate constant k el -the latter is assumed to be the same for ultra-large plus high-molecular weight (UL+HMW) multimers as for low-molecular weight (LMW) multimers. The model is based on the assumptions that (a) HMW and LMW multimers are present in the basal state and/or after DDAVP; (b) UL and HMW multimers can be cleaved to form LMW multimers; (c) we can judge the quantities of UL+HMW+LMW multimers from VWF:Ag measurements; and (d) VWF:CB gives us a measure of the quantity of UL+HMW multimers.

Statistical analysis
Laboratory data were expressed as mean ± standard deviation (SD).
The Student's t-test was used to compare all results, and Pearson's correlation analysis was conducted to assess the association between the RIPA and VWF:Ag parameters. The p-values below .05 were considered statistically significant.

Patients
Seventeen patients with type Vicenza VWD were studied. They  Table 1 shows the patients' main haemostatic findings.

VWF synthesis is normal in type Vicenza VWD
As shown in Table 1

VWF function is normal in type Vicenza VWD
RIPA useful for exploring the interaction between VWF and platelet GPIb, was found sometimes normal, sometimes reduced, even in the same patient (

VWF multimer structure is normal in type Vicenza VWD
Plasma VWF multimer analysis, performed under low-resolution conditions, showed a consistent reduction in VWF levels, with all oligomers present and, in some instances, unusually large VWF multimers as well.

Post-DDAVP multimer behaviour differs from normal in type Vicenza VWD
Post-DDAVP multimer patterns showed an increase in the overall VWF levels, and the appearance of ultra-large VWF multimers (Figure 3), that peaked at 30 min, then the large and ultra-large multimers began to decrease from 60 min onwards. There were no differences in multimer patterns between patients with the p.R1205H mutation alone and those with the p.M740H as well ( Figure 3) Figure 1) or the p.R1205H mutation alone (patient 2 in Figure 1). The pattern is also representative of those seen in the other patients studied . The values are expressed in percentage, taking the total multimers quantity as 100%. Note the relative increase in the representation of LMW multimers in type Vicenza VWD before DDAVP infusion, their rapid decrease 30 min afterwards, and the inverse relationship between high-and low-molecular weight VWF multimers. The latter findings confirm the increased proteolysis of type Vicenza VWF observed by the mathematical model after DDAVP, which does not occur in normal subjects and UL multimers were quantified with a densitometric analysis using the ImageJ software, and expressed as mean ± standard deviation.
The pertinent results are shown in

Mathematical model demonstrates elimination, proteolysis and release of type Vicenza VWF is increased
The metabolic pathways related to VWF after its release from endothelial cells-that is, release, proteolysis and elimination-were studied using the physiology-based mathematical model developed by Galvanin et al. [18]. As shown in Table 3 and Figure 5, the model identified a substantial increase in patients' VWF elimination rate constant (k el parameter, 1.08 −2 /min vs. 1.17 −3 /min in normal subjects), VWF proteolysis rate constant (k proteol , 1.04 −3 /min vs. 4.59 −4 /min in normal) and VWF release rate constant (k rel , 5.87 −2 /min vs. normal 2.74 −2 /min). In other words, all the above pathways seemed to be accelerated, especially the elimination one. The model was also able to quantify the amounts of LMW and UL + HMW multimers before and after DDAVP administration using the VWF:Ag and VWF:CB values. Values of T 1/2 estimated by the model are in good agreement with the observed data post-DDAVP, while the total amount of VWF released (Q) is estimated to be, on average, higher in Vicenza subjects as compared to normal subjects. As seen in Figure 5, the post-DDAVP behaviour of the VWF multimers was characterised by a sudden increase in large and ultra-large multimers and a later less pronounced peak of the LMW forms, which occurred when the large forms were already decreasing. The model also showed that patients' large and small multimers disappeared at the same rate ( Figure 5A).
In contrast, in normal subjects ( Figure 5B) the post-DDAVP increase of large multimers was greater than the one of small multimers, with both oligomers cleared more slowly. Analysing the area under the curve (AUC) of UL+HMW and LMW multimers ( Figure 5C) suggests that in type Vicenza patients, the smaller multimers are derived by proteolysis from the larger ones, explaining why our patients' LMW VWF multimers were relatively more represented than the larger forms, as well as their increased proteolysis rate constant.

DISCUSSION
In this study, we examine whether it is appropriate to classify type Vicenza as a type 1 VWD, considering all of its known in vivo and in vitro features, also with the aid of a mathematical model. We conclude that it would be better to place type Vicenza VWD in a new type 2 class, emphasising the abnormalities peculiar of type Vicenza VWD, and introducing the interaction with clearance receptors as a new functional feature of VWF.
While the phenotype and genotype of type Vicenza VWD have been clearly defined [22], its classification has been subject of different proposals in the last decades, highlighting the difficulty of including type Vicenza in any of the known VWD groups. At present, it is included in type 1 VWD. According to the ISTH [1], type 1 VWD is a partial reduction of VWF without a significantly altered multimer pattern (especially without any loss of large VWF multimers), or any specific abnormalities in the behaviour of VWF towards ligand-binding sites.
This definition does not specify the contribution of VWF synthesis, although we generally assume that a quantitative VWF defect is a consequence of a defective synthesis. It has been suggested that an increase in FVIII:C ratio, as seen in our patients, is indicative of a decreased VWF synthesis [23]. However, several factors go against this conclusion. The normal platelet VWF content and the post-DDAVP normalisation of circulating VWF levels, albeit with short-lived effects or are the consequence of its important increased elimination.
The increased elimination rate of type Vicenza VWF [21,25] is most likely associated with an abnormal interaction between VWF and the binding sites involved in its clearance [26,27]. In this setting, the p.R1205H mutation looks like a gain-of-function mutation that promotes VWF clearance by enhancing its interaction with its receptors [26]. The type Vicenza VWD picture is similar to that of type 2B in which VWF is synthesised and released normally, but its large VWF multimers disappear under the effect of proteolysis due to an increased interaction of VWF with platelet GPIb. It also resembles type 2A-II VWD in which mutations in the A2 domain lead to an increased interaction between VWF and ADAMTS13, with consequent disappearance of large VWF multimers. The key question is whether VWF clearance [25,27], mediated by a number of receptors on macrophages that have yet to be fully identified [28], can be considered one of the VWF functions-like its interaction with platelet GPIb, collagen, FVIII, and other receptors. In conclusion, the above considerations lead us to suggest that it would be better to classify as type 2 VWD all cases of type Vicenza and other type 1 forms involving a shorter VWF survival as the main contributor to the VWD phenotype. To do so, we need to decide whether or not the interaction between VWF and the receptors involved in its clearance is one of the functions of VWF.

AUTHOR CONTRIBUTIONS
A. Casonato designed the research and wrote the paper. E. Galletta performed the haemostatic tests. F. Galvanin performed the mathematical modelling. V. Daidone performed the genetic analysis and analysed the data.