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Keywords:

  • von Willebrand factor, inflammation, cancer, apoptosis, thrombosis.

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

Summary.  von Willebrand factor (VWF) is a protein best known from its critical role in hemostasis. Indeed, any dysfunction of VWF is associated with a severe bleeding tendency known as von Willebrand disease (VWD). Since the first description of the disease by Erich von Willebrand in 1926, remarkable progress has been made with regard to our understanding of the pathogenesis of this disease. The cloning of the gene encoding VWF has allowed numerous breakthroughs, and our knowledge of the epidemiology, genetics and molecular basis of VWD has been rapidly expanding since then. These studies have taught us that VWF is rather unique in terms of its multimeric structure and the unusual mechanisms regulating its participation in the hemostatic process. Moreover, it has become increasingly clear that VWF is a more all-round protein than originally thought, given its involvement in several pathologic processes beyond hemostasis. These include angiogenesis, cell proliferation, inflammation, and tumor cell survival. In the present article, an overview of advances concerning the various structural and functional aspects of VWF will be provided.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

von Willebrand disease (VWD) is a genetic bleeding disorder related to quantitative and/or qualitative abnormalities of von Willebrand factor (VWF). The clinical manifestations of VWD are heterogeneous, and it is therefore not surprising that the classification of VWD has been revised twice since 1987 [1]. Indeed, since the cloning of VWF in the mid-1980s, substantial progress has been made in our understanding of the disease, and a series of excellent reviews have summarized these recent developments (e.g. [2,3]). Large multicenter studies in Europe, Canada and the USA have shed light on the complex genetic background of VWD, particularly of VWD type 1 [2,3]. It is evident that this progress has helped to improve diagnosis, from both a clinical and a laboratory point of view [4,5]. Furthermore, these advances have improved treatment options, and more widespread application of prophylaxis is currently being evaluated [6,7]. Alternative treatment options to the current plasma-derived VWF preparations and desmopressin are being developed, including recombinant VWF and gene therapeutic approaches [8].

As well as clinical studies, animal models of VWD have improved our perception of VWF biology and its potential determinants. This is nicely illustrated by the existence of natural VWD animal models in which the reduced VWF levels are not caused by mutations in the Vwf gene itself, but by mutations in some modifier genes, such as B4galnt2 and St3galIV [9]. These murine studies have found a nice resonance in humans, as a similar complex genetic trait has been recognized for VWD type 1 in humans [2]. Genetically engineered mice have proven to constitute a valuable tool with which to investigate the pathogenesis of VWD. The development of novel techniques to express human VWD-related mutations in VWF-deficient mice now allows detailed in vivo genotype–phenotype analysis. Indeed, the particular phenotypic features associated with VWD types 1, 2A, 2B and 2M have been faithfully reproduced in mouse models [10–14].

The combination of population-based studies and the expanding availability of animal models is leading to constant improvement in our approach to VWD. However, the equation is not complete without a closer look at the protein behind VWD, and the recent discovery of unknown functional and structural aspects of VWF may also contribute to a broadening of our perspective on VWD. This is why the remainder of this review will focus not so much on VWD, but rather on new insights into the old functions of VWF and on the discovery of functional aspects that lie beyond hemostasis.

VWF structure: old and new come together

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

The cloning of the VWF cDNA in the mid-1980s was an important step forwards in discovering the architecture of the VWF domain structure (reviewed in [15]). Five different domain structures could be distinguished on the basis of sequence homology alignment, the arrangement of which is illustrated in Fig. 1A. This domain structure has been used throughout the literature since then. However, the structures of many more proteins containing homologous domains have been solved in the last two decades. Recently, Zhou et al. have re-evaluated the VWF domain structure, using the updated information on the structures of these homologous domains in combination with state-of-the-art electron microscopy techniques [16–18]. This analysis not only allowed a more detailed assignment of the disulfide bonds between cysteines, but also resulted in a redesign of the VWF domain structure (Fig. 1B) [18]. In particular, the C-terminal part following the D4 domain was previously thought to comprise three B-domains, two C-domains, and one cysteine knot (CK) domain (Fig. 1A). According to the analysis by Zhou et al., the region separating the D4 and CK domains contains six consecutive C-domains, with the Arg-Gly-Asp motif being located in the C4 domain (Fig. 1B). Zhou et al. [17] also used electron microscopy techniques to obtain a better look at the structural organization of the VWF molecule. Using this approach, they observed that, under slightly acidic conditions, a dimeric fragment containing the A3-CK region assembles into an intertwined bouquet structure in which the C domains separate the A3-D4 region and the CK domain, like the stem of a flower (Fig. 1B).

image

Figure 1.  Domain structure of von Willebrand factor (VWF). The cloning of VWF in the 1980s provided the first insights into the structural organization of the protein, and a domain arrangement was proposed based on internal sequence homology (reviewed in [15]). This domain structure is schematically depicted in (A), and has generally been used throughout the literature in the last three decades. By using updated information on the structures of homologous domains in other proteins, Zhou et al. [18] re-evaluated the VWF domain structure. This analysis resulted in a redesign of the VWF domain architecture, shown in (B). The same group also used negative stain electron microscopy to visualize the VWF structure, and showed that the dimeric VWF assembles into an intertwined bouquet structure under slightly acidic conditions [16–18].

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High-resolution microscopic techniques have also been used to monitor the overall structure of VWF, with a focus on eventual conformational changes in the protein that occur when it is exposed to increased shear stress [19–21]. These studies revealed that VWF circulates in a globular form under conditions of low shear stress, but changes into an elongated form under the influence of increased hydrodynamic shear forces. These structural changes are crucial to the nature and function of VWF, and two well-known examples are the interaction with platelets, and proteolysis by ADAMTS-13 [16,21,22]. Interestingly, recent studies have revealed that exposure to shear stress has a number of other important effects on VWF. First, exposure of VWF to shear stress provokes a rearrangement of disulfide bridges in the C-terminal part of the protein [23,24], a process that contributes to the incorporation of new VWF molecules into the assembled VWF strings at the vascular surface [23,24]. It is of note that this shear stress-induced disulfide rearrangement can be modulated by a previously unrecognized function of ADAMTS-13, i.e. a thiol reductase activity that is located in the C-terminal region of ADAMTS-13 [25]. Second, elongated VWF is much more susceptible to methionine oxidation in the A domains in the presence of reactive oxygen species [26]. Following oxidation of several methionines, the functional properties of VWF are changed importantly: VWF is more efficient in its interaction with platelets, and oxidation renders the protein resistant to ADAMTS-13-mediated proteolysis [26–28]. Finally, two groups simultaneously reported a role for shear stress in VWF clearance. Exposure to shear stress converts VWF into a ligand for the receptor LDL receptor-related protein 1, resulting in increased in vitro and in vivo uptake of VWF by macrophages, cells that predominate in the clearance of VWF [29,30].

To conclude on this aspect, and looking back at the progress made in the last 2–3 years, there has been a surge of new and exciting data on the structure and the shear-dependent regulation of VWF. If and how our knowledge about the various functional properties of the protein will need to be adapted in the light of these newly identified structural features remains to be determined. This is where the ‘unknown’ part of our title comes into play, leading to promises of additional years of VWF research ahead of us.

VWF function in thrombosis: new tricks for an old protein

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

The historical connection between VWF and atherothrombotic complications originates from its capacity to promote platelet adhesion at high shear stress. In contrast, the link between VWF and venous thrombosis was long considered to be indirect and related to coagulation factor VIII [31]: VWF functions as a carrier protein for FVIII, and there is a strong correlation between the levels of FVIII and those of VWF [32,33]. Thus, high levels of VWF lead to high levels of FVIII, which, in turn, create a higher risk of venous thrombotic complications [34]. This scenario is supported by genetic studies (for instance, the VWF rs1063856 single-nucleotide polymorphism in exon 8 is associated with an increased venous thrombotic risk [35]). Moreover, Wetzstein et al. [36] reported a case of suspected sinus venous thrombosis in a newborn with VWD type 3, suggesting that venous thrombosis may occur in the absence of VWF. On the other hand, there is accumulating evidence that VWF itself plays an active role in the development of venous thrombi. VWF was identified as an independent risk factor in the LITE study, which was a prospective study aimed at identifying markers for venous thromboembolism [37]. Furthermore, immunohistochemical analysis revealed the presence of VWF in thrombi of patients who died from venous thrombotic complications [38]. Animal models are also in support of a role for VWF in venous thrombosis, as the inhibition or genetic deficiency of VWF protects against venous occlusion in various experimental models [39–41]. The mechanism by which VWF contributes to the formation of venous thrombi is only partially understood. Intriguingly, Chauhan et al. [40] found that platelet recruitment by VWF does not solely rely on interactions between VWF and glycoprotein (GP)Ibα, but that, under venous shear conditions, VWF may also recruit platelets via other, so far unidentified, platelet receptors. Moreover, recent findings have also put another player into the equation for venous thrombus formation: extracellular DNA traps (so-called neutrophil extracellular traps [NETs]) [42,43]. These NETs are formed upon vascular injury, and consist of a meshwork of DNA fibers comprising citrullated histones that promote the prothrombotic capacity of these NETs (for a detailed review, see [43]). NETs are also able to catch several adhesive plasma proteins, including VWF, which further enhance the recruitment of platelets, and NETs were indeed seen to colocalize with VWF in venous thrombi [42,44].

VWF has also been found to be associated with ischemic stroke (for a review, see [45]). It should be noted that epidemiologic studies have been ambiguous on the relationship between VWF and risk of cerebral occlusive complications [45,46]. In contrast, experimental mouse models of stroke have shown that the absence of VWF protects the mice from brain ischemia/reperfusion injury [47,48].

These new findings clearly show that VWF plays a role beyond atherothrombosis, and identify it as a critical component for venous thrombosis and ischemic stroke (Fig. 2). Targeting of VWF could therefore represent a viable option for the treatment and/or prevention of these vaso-occlusive complications.

image

Figure 2.  Functional diversity of von Willebrand factor (VWF). VWF is best known for its role in hemostasis, with particular reference to the recruitment of platelets under conditions of arterial shear stress and its carrier function for coagulation factor  VIII. However, it is becoming increasingly clear that VWF functions extend much further than that. In this figure, we have illustrated the functional diversity of the protein, highlighting its involvement in arterial and venous thrombosis, angiogenesis, smooth muscle cell proliferation, inflammatory processes, and apoptosis. In addition, rather than acting as a circulating chaperone for FVIII alone, VWF has been found to be circulating in complexes with several other proteins as well. Therefore, VWF can be considered to be a molecular bus.

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VWF and inflammation

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

Similarly to the situation for venous thrombosis, the link between VWF and inflammatory processes was initially thought to be indirect. This was based on the observation that VWF is responsible for the targeting of P-selectin to the Weibel–Palade bodies, which is a necessary step in the process leading to the regulated exposure of P-selectin at the surface of the stimulated endothelial cell [49]. Deficiency of VWF provokes impaired P-selectin surface expression and subsequent defects in leukocyte recruitment in the early phases of inflammation [50]. However, there are now also indications that VWF may actively participate in the inflammatory response (Fig. 2). First, VWF can function as an adhesive surface for leukocytes via interactions with P-selectin GP ligand-1 and β2-integrins [51]. Recently, the immune cell receptor Siglec-5 has also been identified as a receptor for VWF [52]. Second, platelet-decorated VWF strings at the surface of endothelial cells efficiently recruit leukocytes, even under conditions of high shear stress [53]. Finally, VWF–platelet complexes are critical to optimal extravasation of leukocytes [54].

It is important to keep in mind that our knowledge of the physiologic relevance of VWF in inflammatory processes is primarily derived from animal studies using different models for inflammation, such as atherosclerosis, wound healing, experimental allergic encephalomyelitis, cytokine-induced meningitis, and stroke [48,50,55,56]. In patients, this connection is less well established, which is probably attributable to the multifactorial nature of these inflammatory conditions. An example of such a dichotomy between animal and human studies is atherosclerosis. Indeed, VWF-deficient pigs and VWF-deficient mice develop fewer atherosclerotic lesions, pointing to a role of VWF in the recruitment of leukocytes to lesion sites [55,57]. However, human studies have failed to confirm that VWF deficiency is protective against the development of atherosclerosis (for a recent review, see [58]). Of course, in contrast to the VWF-deficient mice, most of the VWD type 3 patients receive VWF replacement therapy, which replenishes the reservoir of circulating VWF on a regular basis.

The various animal models have also provided insights into how the contribution of VWF to the inflammatory process might be regulated. First, regulatory secretion of VWF from endothelial cells and/or platelets allows the protein to participate in a timely manner in the inflammatory response process [50,54,59]. On the other hand, how can the proinflammatory activity of VWF be counteracted? It appears that the VWF-cleaving protease ADAMTS-13 plays an important role in this regard: deficiency of ADAMTS-13 is associated with increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins, but only selectively when VWF is present [60]. This regulatory role of ADAMTS-13 was confirmed in experimental models of atherosclerosis, in which the lack of ADAMTS-13 exaggerates the VWF–platelet-dependent inflammatory response, manifested by increased leukocyte recruitment to lesion sites [61]. Also in the experimental stroke models, ADAMTS-13 plays an important role in regulating VWF-dependent inflammatory responses. Whereas ADAMTS-13 deficiency increases susceptibility to focal cerebral ischemia [48,62,63], intravenous administration of recombinant ADAMTS-13 in wild-type mice markedly reduced infarction volume [48]. Thus, proteolytic degradation of VWF by ADAMTS-13 downregulates the proinflammatory potential of VWF. Apart from ADAMTS-13, VWF may also be degraded by proteases released from leukocytes [64–66].

In conclusion, VWF is tightly associated with the inflammatory response in both a direct and an indirect manner, and several regulatory pathways have been developed to prevent excessive VWF-dependent inflammation. As such, VWF may serve as a therapeutic target to downregulate inflammation under pathologic conditions such as stroke.

VWF as a molecular bus

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

As mentioned above, the 250-kDa subunit structure of VWF contains four different types of domain according to the annotation by Zhou (Fig. 1B) [18], each of them being characterized by its specific type of folding. This architectural diversity provides VWF with the potential to interact with a wide spectrum of structures, and, indeed, a large number of protein and non-protein ligands for VWF have been identified. A list of 24 different protein ligands that originate from the human proteome is provided in Table 1. Given the biodistribution of VWF, there are many locations where VWF can meet and interact with these ligands, and it is important that each of these interactions occurs in a timely manner. For instance, mature VWF tightly interacts with its propeptide within the Weibel–Palade bodies, which is needed for optimal multimerization of the protein and the formation of a functional FVIII-binding site [15,67,68]. This interaction is highly pH-dependent, and is most efficient under the slightly acidic conditions that are present inside the Weibel–Palade bodies. Upon secretion, the complex is exposed to higher pH, promoting its rapid dissociation [69]. Complex dissociation is needed because the presence of the propeptide may (i) attenuate VWF-dependent platelet activation and adhesion [70], and (ii) impair recruitment of FVIII to the D′D3 domain [71].

Table 1.   Human protein ligands for mature von Willebrand factor (VWF)
NameBinding domainSite of interactionReference
  1. CK, cysteine knot; CTGF/CCN2, connective tissue growth factor; IGFBP7, insulin-like growth factor binding protein-7; LRP1, LDL receptor-related protein 1; PSGL-1, P-selectin glycoprotein ligand-1.

VWF propeptideD′D3Endothelial cells; platelets[97]
FVIIID′D3Circulation; some endothelial cells[32]
P-selectinD′D3Endothelial cells[98]
β2-IntegrinsD′D3, A1, A2, A3Leukocytes[51]
Glycoprotein IbαA1Platelets[99]
OsteoprotegrinA1Circulation; endothelial cells[73]
β2-Glycoprotein IA1Circulation[100]
PSGL-1A1Leukocytes[51]
Collagen VIA1Subendothelial matrix[101]
ADAMTS-13A1, A2, A3, D4Circulation[72]
Collagen IA3Subendothelial matrix[102]
Collagen IIIA3Subendothelial matrix[102]
ThrombospondinA3Circulation[103]
IGFBP7D4-CKCirculation; endothelial cells[84]
αIIbβ3C4 (RGD motif)Platelets[104]
αvβ3C4 (RGD motif)Endothelial and tumor cells[104]
FibrinC1–6Thrombus[105]
CTGF/CCN2CKEndothelial cells[83]
Galectin-1Glycan moietiesCirculation; endothelial cells[74]
Galectin-3Glycan moietiesCirculation; endothelial cells[74]
Siglec-5Glycan moietiesMacrophages[52]
Angiopoietin-2UnknownCirculation; endothelial cells[82]
Interleukin-8UnknownEndothelial cells[106]
LRP1UnknownMacrophages[29]

The ability of VWF to associate with FVIII in the circulation is the first and best characterized example of VWF function as a carrier protein. The recent discovery that other proteins are circulating together with VWF led us to propose the notion of VWF as a molecular bus (Fig. 2). Other proteins that are associated with VWF in the circulation include ADAMTS-13 [72], osteoprotegerin [73], angiopoietin-2 (Christophe OD, Cherel G, Lenting PJ, Denis CV; unpublished observations), and two members of the galectin family, galectin-1 and galectin-3 [74]. The reasons why proteins associate with VWF may be different. We know that binding of FVIII to VWF is needed to maintain appropriate plasma levels, as FVIII plasma levels and half-life are remarkably reduced in the absence of VWF [32]. But is this also true for the other ligands? So far, this is not completely clear. For angiopoietin-2 and osteoprotegerin, we have been unable to retrieve any information in this regard. In contrast, we observed that galectin-1 and galectin-3 levels were significantly reduced in VWF-deficient mice [74], suggesting that VWF may protect these proteins in the circulation, similarly to FVIII. Surprisingly, the opposite seems to be the case for ADAMTS-13. Mannucci et al. observed an inverse relationship between plasma levels of ADAMTS-13 and of VWF [75]. Moreover, ADAMTS-13 levels were ∼ 40% higher in VWD type 3 patients than in sex-matched and age-matched healthy controls [75]. Given that the half-life of ADAMTS-13 is longer than that of VWF [76], it is possible that the VWF-bound ADAMTS-13 fraction is cleared more rapidly, contributing to the lower levels of ADAMTS-13 in the presence of VWF.

Another reason why proteins associate with VWF may be related to their targeting. It has frequently been mentioned that VWF targets FVIII to sites of injury. This is an attractive option from a mechanistic point of view; however, no specific studies providing evidence for this possibility have been published. In truth, this might be experimentally challenging to demonstrate, as probably < 10% of the circulating VWF multimers are actually carrying an FVIII molecule. Perhaps easier to address is the complementary question: can FVIII target the injured vessel wall in the absence of VWF? Answering this question is of relevance for the development of FVIII variants that do not rely on VWF to survive in the circulation. Chauhan et al. [40] elegantly provided a first indication in this regard. They showed that infusion of recombinant FVIII into VWF-deficient mice resulted in strongly reduced embolism in a ferric chloride-induced thrombosis model. Thus, the presence of FVIII was able to stabilize the developing thrombus, indicating that FVIII arrived at the site of injury in the absence of VWF. It is of note that the presence of FVIII was insufficient to promote full occlusion of the vessels. Holmberg et al. have recently confirmed this important finding, using an alternative approach [77]. Whether VWF also targets its other ligands to their destination has not yet been investigated. With regard to ADAMTS-13, it has been speculated that binding to VWF would facilitate its immediate action to proteolyse the active platelet-binding VWF conformation in order to prevent excessive thrombus formation at the site of injury [72].

Complex formation may affect not only plasma levels or targeting, but also the function of VWF and/or its ligands. It has been well established that VWF-bound FVIII is unable to interact with components of the tenase complex (for a review, see [32]), so VWF clearly influences FVIII function. The opposite is also true, as FVIII may also modulate VWF function, albeit in an indirect manner. In the presence of FVIII, VWF appears to be more susceptible to proteolysis by ADAMTS-13 [78], thereby reducing the hemostatic potential of VWF. The in vivo relevance of this FVIII-mediated modulation has not yet been determined. However, in view of the possibility that > 90% of VWF is FVIII-free, the effect of FVIII on ADAMTS-13-mediated proteolysis is probably limited to a small proportion of the VWF population.

With regard to the other proteins associated with VWF, their mutual effects on each others’ functions has been only sparsely investigated, with the exception of ADAMTS-13. ADAMTS-13 is an important regulator of VWF multimer size, thereby determining its hemostatic (and proinflammatory) potential [72]. Regarding the galectins, we have recently observed that the presence of galectins prevents excessive VWF multimer assembly at the endothelial cell surface in vitro and in vivo [74]. However, no information is available on whether VWF modulates the function of galectins, which participate in various pathways associated with cancer development, inflammation, and apoptosis (for reviews, see [79,80]). Even less is known about the effects of VWF on the functions of osteoprotegrin and angiopoietin-2 and vice versa. Considering the important roles of these proteins in bone metabolism and vascular maturation, respectively, it would be most interesting to obtain more insights into this matter. Thus, many unknowns remain regarding why such proteins circulate together with VWF and how their association may regulate each others’ functions.

VWF as a cell effector: angiogenesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

The long list of VWF ligands contains a number of proteins that are involved in cellular signaling processes. The archetype of VWF-mediated signaling relates to its interaction with its platelet receptor GPIbα, which initiates transmembrane signaling events that ultimately result in αIIbβ3 activation and platelet aggregation (for a review, see [81]). In this part of the review, we will provide a number of examples that show that the cell effector functions of VWF are not limited to platelet activation, but are actually more widespread than previously supposed. Moreover, they may directly modulate various (patho)physiologic processes (Fig. 2).

First, Starke et al. demonstrated that the lack of VWF promotes angiogenic processes, as was illustrated by markedly increased endothelial cell proliferation in the absence of VWF in vitro and an increased vessel density of the vasculature in the ears of VWF-deficient mice [82]. The molecular basis of this VWF-dependent effect is still not completely clear. Experimental data point to VWF as a negative modulator of vascular endothelial growth factor (VEGF)-dependent angiogenesis via multiple intracellular and extracellular pathways involving αvβ3 and angiopoietin-2, both of which are ligands for VWF (Table 1) [82]. However, other pathways may also be involved. For instance, both galectin-1 and galectin-3 have been found to be proangiogenic, particularly with regard to tumor angiogenesis [80]. As at least part of the galectin population is associated with VWF inside and outside the endothelium [74], the possibility exists that VWF modulates angiogenesis via these galectins as well. Furthermore, two other angiogenic regulators that are expressed in endothelial cells have recently been identified as binding partners for VWF: connective tissue growth factor and insulin-like growth factor binding protein-7 [83,84].

Although it remains to be determined whether the modulating effect of VWF on angiogenesis is qualitative or quantitative, it seems reasonable to assume that its role is somehow linked to the angiodysplasia that is observed in a subset of VWD patients. Angiodysplasia may originate from vascular malformations resulting from an impaired angiogenesis process [85]. The bleeding complications associated with angiodysplasia have been recognized as a severe complication in VWD [86]. Most frequently, it is observed in patients lacking high VWF multimers because of either hereditary defects (VWD types 2A and 3) [86,87] or acquired conditions (Heyde’s syndrome and patients carrying circulatory assist devices) [88,89]. The reason why the lack of high VWF multimers particularly predisposes to an increased risk of vascular malformation is unknown, and merits further study.

VWF as a cell effector: cell proliferation

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

In the previous section, we have seen that the presence of VWF in endothelial cells limits VEGF-induced cell proliferation. Surprisingly, opposite effects have been observed with regard to other cells. Following vascular injury, VWF may penetrate into the intima of large peripheral vessels, where it comes into contact with smooth muscle cells [90]. Deposition of VWF in the intima is associated with intimal thickening [91], suggesting that VWF contributes to the proliferation of cells. In vitro studies have revealed that VWF directly stimulates smooth muscle cell proliferation, providing a rationale for the observed hyperplasia [91]. More recently, it was found that the mitogenic effect of VWF involves prompt and robust upregulation of multiple genes associated with growth factor stimulation [92]. The physiologic relevance of this mitogenic activity of VWF is nicely illustrated in a disorder known as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, a rare genetic disorder associated with recurrent strokes and dementia [92]. VWF is abundant in the brain vessels of these patients, the subarachnoid arteries of which are characterized by concentric thickening of the media and adventitia [92]. It is tempting to speculate that VWF plays an active role in the thickening of the vascular wall in this disease.

VWF as a cell effector: apoptosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

Besides modulating cell proliferative processes, VWF may also have some more devastating effects. Recently, it was reported that platelet apoptosis could be induced via the VWF–GPIbα axis, in a pathway involving caspase-3, Bak, and Bax [93]. This proapoptotic effect may be of particular relevance for those pathologic situations where unwanted VWF–GPIbα interactions take place, e.g. in VWD type 2B, which is manifested by continuous interactions between type 2B VWF mutants and GPIbα. Indeed, preliminary experiments using our mouse model for VWD type 2B indicate that at least part of the platelet population in these mice is apoptotic, which could explain, in part, the thrombocytopenia observed in these mice. However, additional studies are needed in this regard.

The potential proapoptotic properties of VWF are not restricted to platelets. Previously, we have observed that tumor cells have a higher metastatic potential in VWF-deficient mice than in VWF-expressing mice [94]. Preliminary studies suggested that VWF reduced metastasis by inducing cell death of tumor cells [94,95]. However, it was a recent study by Mochizuki that elegantly unraveled the mechanism that explained the increased metastasis in the absence of VWF. First, the authors confirmed that VWF displays a proapoptotic effect towards tumor cells, probably via the interaction with αvβ3 [96]. Second, they demonstrated that the metastatic capacity of tumor cells correlates with the expression levels of a protease named ADAM-28 [96]. Unexpectedly, VWF appeared to be a preferred substrate for this protease. The authors found that ADAM-28 cleaves human VWF at two distinct sites in a shear stress-independent manner, thereby destroying the proapoptotic function of VWF. Thus, VWF serves as an inhibitor of tumor cell survival, and certain tumor cells have armed themselves against VWF via the production of ADAM-28.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

Despite the undeniable progress made in our knowledge of VWF structure and function in the previous three decades, it appears that we still have only a glimpse of the functional diversity of this intriguing protein. It was tempting to think that at least its classic role in hemostasis was largely understood, but new developments with regard to its involvement in venous thrombosis and stroke have proven this assumption to be untrue. In fact, many aspects of VWF biology remain to be explored, with particular reference to its cell effector functions and its role as a molecular bus, suggesting that VWF-related research has a bright future ahead.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References

Many interesting and relevant reports and reviews have been published on the various aspects of VWF. Unfortunately, we could incorporate only a fraction of them in this review, owing to size restrictions, and we apologize to those authors whose papers could not be referenced.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. VWF structure: old and new come together
  5. VWF function in thrombosis: new tricks for an old protein
  6. VWF and inflammation
  7. VWF as a molecular bus
  8. VWF as a cell effector: angiogenesis
  9. VWF as a cell effector: cell proliferation
  10. VWF as a cell effector: apoptosis
  11. Concluding remarks
  12. Addendum
  13. Acknowledgements
  14. Disclosure of Conflict of Interests
  15. References
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