Clearance of von Willebrand Factor

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Clearance of von Willebrand Factor

Cécile V. Denis 1, Olivier D. Christophe1, Beatrijs D. Oortwijn2, Peter J. Lenting 2,3

1INSERM U770, Le Kremlin-Bicêtre, F-94276, France; Univ Paris-Sud, Le Kremlin-Bicêtre, F-94276, France

2Laboratory for Thrombosis and Haemostasis, Department of Clinical Chemistry & Haematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands

3Crucell Holland B.V., Department of Protein Discovery, Archimedesweg 4-6, 2333 CN Leiden, the Netherlands

Running title: Clearance of von Willebrand factor

Word count:

Abstract: 201

Text: 4496

Address correspondence to:

Dr. C.V. Denis


80 rue du Général Leclerc

94276 Le Kremlin-Bicetre Cedex

Tel: 33-(0)1-49-59-56-05

Fax: 33-(0)1-46-71-94-72



The life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of the VWF gene in endothelial cells and megakaryocytes to the removal of VWF from the circulation. The various aspects of VWF clearance have been the objects of intense research in the last few years, stimulated by observations that VWF clearance is a relatively common component of the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, improving the survival of VWF is now considered as a viable therapeutic strategy to prolong the half-life of factor VIII in order to optimise treatment of haemophilia A.

The present review aims to provide an overview of recent findings with regard to the molecular basis of VWF clearance. A number of parameters have been identified that influence VWF clearance, including its glycosylation profile and a number of VWF missense mutations. In addition, in vivo studies have been used to identify cells that contribute to the catabolism of VWF, providing a starting point for the identification of receptors that mediate the cellular uptake of VWF. Finally, we discuss recent data describing chemically modification of VWF as an approach to prolong the half-life of the VWF/FVIII complex.


For each organism, the regulation of protein levels is a key process that requires a delicate balance between biosynthesis and elimination. Defects in clearance mechanisms may result in deficiency or an accumulation of certain components, which may eventually provoke pathological manifestations. For instance, the accumulation of amyloid  peptide due to reduced LRP-mediated clearance contributes to the pathogenesis of Alzheimer disease1,2, whereas defects in the clearance of lipoprotein particles are associated with familial hypercholesterolemia.3 These two examples illustrate the universal requirement for mechanisms that control the clearance of proteins, and the haemostatic system is not an exception in this regard.

One important player of the haemostatic system is von Willebrand factor (VWF). VWF is a multimeric protein which contributes to the initial recruitment of platelets to injured vessel by acting as a molecular bridge between the exposed subendothelial matrix and the platelet receptors GpIb/IX/V and αIIbβ3.4 Apart from its role in primary haemostasis, a number of other functions have been recognized as well. First, VWF and its propeptide are indispensable for the intracellular formation of endothelial-specific storage organelles, the Weibel-Palade bodies.5,6 These organelles are not only a storage-compartment for ultra-large VWF multimers, but also for a variety of other proteins, such as P-selectin, interleukin-8 and angiopoietin-2 (for reviews see Michaux et al. and Rondaij et al.).7,8 Second, the presence of VWF has a suppressing effect on the metastatic potential of tumour cells.9 Third, VWF comprises binding sites for Staphylococcal aureus-surface proteins (Protein A), which may facilitate intravascular colonisation by these pathogens.10-12 Further, VWF may promote the proliferation of smooth muscle cells.13 Finally, we have recently reported on the ability of VWF to act as an adhesive surface for leukocytes.14 VWF-leukocyte interactions involve P-selectin glycoprotein ligand 1 (PSGL-1) as well as the family of 2-integrins.

Another intriguing aspect of VWF is that it functions as a carrier to transport other proteins in the circulation. A recent example of a molecule that uses VWF as a molecular vehicle is osteoprotegerin. This protein is co-expressed with VWF in endothelial cells, and upon secretion both proteins remain in complex.15,16 The physiological relevance of complex formation between VWF and osteoprotegerin is yet unknown and remains to be resolved. The necessity for complex formation with VWF is much clearer for coagulation factor VIII (FVIII). FVIII is a plasma protein that functions as a cofactor for activated factor IX, and functional deficiency of FVIII leads to impaired generation of activated factor X.17 This deficiency is associated with a severe bleeding tendency, known as haemophilia A.18 Patients lacking VWF are characterized by a secondary deficiency of FVIII 19, demonstrating that VWF is essential for appropriate survival of FVIII.

The present review aims to summarize recent developments regarding clearance of VWF. Knowledge of the mechanisms that are responsible for the clearance of VWF complex is important, because modulated clearance of VWF has been found to contribute to the pathogenesis of certain forms of von Willebrand disease (VWD). Moreover, since VWF is critical for the survival of FVIII, manipulating VWF clearance may be an approach to improve survival of FVIII in order to reduce the treatment-frequency of haemophilia A patients.

Biosynthesis of VWF

VWF is produced in endothelial cells and megakaryocytes.20,21 Megakaryocytic production is responsible for the presence of VWF in the -granules of platelets, whereas endothelial cells are the primary source of VWF that is found in the subendothelial matrix and in plasma. Biosynthesis generates a single-chain pre-pro-polypeptide of 2813 amino acids, in which a number of domainal structures can be distinguished: D1-D2-D’-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK.19 After removal of the signal peptide, pro-VWF subunits undergo tail-to-tail linkage via disulphide bonding within the cysteine-rich CK-domains, resulting in pro-VWF dimers. Further processing proceeds within the Golgi-apparatus and involves multimerization via intramolecular cysteine-bonding within the D’-D3 domains. This process produces a heterogeneous pool of differentially sized multimers, with molecular masses ranging from 0.5x106 to over 10x106 Da. Imperative to this multimerization is binding of propeptide to the D’-D3 region, an interaction which is optimal under slightly acidic conditions such as those present in the Golgi.20,22 Indeed, defective interactions between propeptide and D’-D3 domains lead to impaired multimerization.23 Of interest, the slightly acidic conditions needed for optimal propeptide binding also apply to the binding of proteins that co-localize with VWF in the endothelial storage organelles, such as osteoprotegerin and IL-8.16,24

Finally, limited proteolysis in the trans-Golgi network separates the propeptide from the mature VWF multimer. Proteolysis is believed to be mediated by furin, which recognizes a positively-charged sequence downstream the Arg763-Ser764 cleavage site.25 Mutations in this area lead to incomplete removal of the propeptide, thereby generating pro-VWF multimers displaying suboptimal FVIII binding.26,27 Following proteolysis, VWF is targeted to the storage organelles. Noteworthy, both VWF and propeptide are absolutely necessary for Weibel-Palade body formation, whereas -granule formation occurs in their absence. Endothelial cells obtained from animals with a complete VWF-deficiency indeed lack Weibel-Palade bodies 5,28, and the formation of these storage-organelles can be restored upon transfection with VWF.5 The importance of VWF for Weibel-Palade body formation is further underscored by the fact that mutations associated with impaired multimerization also affect size and number of Weibel-Palade bodies.7

Glycosylation of VWF

Apart from multimerization and proteolytic processing, VWF is also subject to extensive glycosylation. The amino acid sequence of VWF predicts the site of 12 N-linked and 10 O-linked oligosaccharide side-chains, and this extensive number of glycans contributes to almost 20 % of the molecular mass of the VWF molecule.29 N-linked glycosylation is initiated within the endoplasmatic reticulum, and this process continues within the Golgi apparatus. N-linked glycans on VWF display a wide spectrum of complexity, and include bi- tri- and tetra-antennary structures.30 Interestingly, VWF is one of the few plasma proteins that have blood group A, B and H antigenic structures incorporated in their oligosaccharide side-chains.31 It is important to stress that the blood group determinants are attached to the N-linked but not the O-linked structures.32 As for the O-linked glycans, the majority consists of sialylated T-antigen, a mucin-type core 1 structure.32 The contribution of glycosylation to VWF biology is still a poorly understood area, despite many years of research. It seems conceivable to assume that optimal glycosylation is required for proper synthesis and secretion. Furthermore, the presence of the glycan structures also seems to be involved in regulating VWF function, an issue that is discussed in more detail elsewhere.33,34 Finally, the glycosylation profile of VWF has proven to be a prominent parameter that influences VWF plasma levels.

Glycosylation of VWF: effect of blood group determinants on VWF levels

More than two decades ago, it was already recognized that VWF levels are lower in individuals having blood group O compared to those having non-O 35, and this observation has been confirmed in many other studies (reviewed in 34). On average, VWF concentrations are approximately 25% lower in persons with blood group O than those in non-O individuals. Levels are even more reduced in persons with the Bombay-phenotype, who lack expression of ABO-antigens.36

How to explain the lower levels of VWF in those who have blood group O? Studies by O’Donnell and colleagues have provided evidence that the effect is mediated by the ABH antigenic structures present on the VWF molecule self rather than via indirect mechanisms.37 They found a correlation between expression levels of A-transferase, the amount of A antigenic determinants present on the VWF molecule and corresponding VWF levels. A similar relationship between the loading of VWF with A or B determinants and VWF levels has more recently been reported by Morelli and colleagues.38 Thus, in some way the blood group determinants present on the VWF molecule affect biosynthesis, secretion, clearance or a combination thereof.

Attempts to address the effect of blood group determinants on biosynthesis and secretion in vitro have been proven to be difficult, because of the absence of A- and B-transferase activity in isolated human umbilical vein endothelial cells, the main primary cell type that is used to study VWF biology.39,40 However, a number of arguments are available that point against a major effect of the blood group determinants on biosynthesis and/or secretion. First, VWF concentrations in platelets are affected to a minor extent by blood group type 41, indicating that the blood group determinants do not affect synthesis and storage in megakaryocytes. Second, the rise in VWF levels upon desmopressin treatment appears to be similar for patients with different blood groups 42, suggesting similar efficiency of VWF secretion from endothelial storage pools.

Based on these observations, it seems conceivable that the effect of blood group determinants is most likely affecting clearance of VWF. Although not formally proven, there are a number of observations that are in support of this possibility. First, preliminary data were recently presented concerning the half-life of VWF following desmopressin treatment in healthy individuals.43 It was found that that the half-lives were significantly shorter in O-subjects than in non-O-subjects. Another observation relates to the ratio between VWF propeptide and mature VWF. VWF propeptide and mature VWF are released simultaneously in a 1:1 molar ratio. However, propeptide is cleared 3- to 4-fold more rapidly than VWF, resulting in distinct propeptide/VWF ratio under steady-state conditions.44 Interestingly, this ratio is dissimilar between individuals with different blood groups, in that blood group O individuals have an increased ratio compared to non-O persons.45,46 Given the notions that (1) VWF and propeptide are released in a 1:1 ratio and (2) propeptide levels are unaffected by blood group, it should follow that VWF has a shorter half-life in blood group O persons. This possibility has been addressed by Nossent et al. using a model assuming that the propeptide half-life is more or less constant amongst individuals. This model predicts the half-life of endogenous VWF to be 2 h longer in non-O persons compared to blood group O persons.46 Finally, several studies have reported that the half-life of infused FVIII correlates with steady-state VWF levels, its carrier protein.47,48 When we analysed this correlation in a cohort of 38 haemophilia A patients, we found that this correlation is strongly influenced by blood group (Fig. 1). This may explain that why the half-life of FVIII (irrespective whether it is recombinant or plasma-derived), is considerably shorter in haemophilia A patients with blood group O with FVIII half-lives being 11.52.6 h and 14.33.0 h (p = 0.0044) in O- and non-O patients, respectively. A similar effect of blood group on FVIII survival has previously been reported.48 Since no blood group determinants are present on the FVIII molecule, the most logical explanation for the blood-group dependent half-life of FVIIII is that the half-life of its carrier protein VWF is determined by the blood group determinants.

The notion that ABO-determinants influence VWF clearance may be of relevance for the development of VWF preparations for therapeutic use, for instance by increasing the amounts of non-O VWF molecules in plasma-derived VWF concentrates. Moreover, in view of the development of a recombinant-derived VWF therapeutic preparation 49, it is of importance to investigate how the non-human glycosylation profile affects the survival of VWF in humans. In particular, it is of relevance to exclude that the glycan profile of recombinant VWF mimics that of the Bombay-phenotype, since this phenotype is associated with low VWF levels.36

Apart from the ABO-blood group effect, other blood group-dependent modifications of the glycosylation profile may also affect VWF plasma levels. An example hereof is the Secretor system, which is involved in the ability to secrete blood type antigens in body fluids. Persons with blood group O and the SeSe-genotype have significantly higher VWF levels (approximately 20%) compared to those with the O-blood group combined with the Sese- or sese-genotype.50 It is not known whether Secretor-dependent modifications affect VWF clearance, and propeptide-analysis would provide insight in this regard.

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