Structure and function of von Willebrand factor

Hassan, Md. Imtaiyaz; Saxena, Aditya; Ahmad, Faizan

Blood Coagulation & Fibrinolysis: January 2012 - Volume 23 - Issue 1 - p 11–22
doi: 10.1097/MBC.0b013e32834cb35d

von Willebrand factor (VWF) is a long plasma protein that contains many domains and each domain has its own function. VWF exists in a multimeric form and performs varieties of functions in the human body, including thrombus formation and blood coagulation. The crystal structures of three subdomains are known, and, interestingly, all three domains share identical three-dimensional fold with α−β−α sandwiched model. VWF is directly associated with different types of von Willebrand disease. In this review, our aim is to gather recent developments on structure and functions of VWF and its clinical relevance.

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India

Correspondence to Md. Imtaiyaz Hassan, PhD, Assistant Professor, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110 025, India Tel: +91 11 2698 1733; fax: +91 11 2698 3409; e-mail:

Received 5 April, 2011

Revised 2 June, 2011

Accepted 1 September, 2011

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von Willebrand factor (VWF is a long polypeptide chain having a molecular mass of ∼270 kDa. This multimeric plasma protein is present in blood plasma and platelets, and is produced constitutively in endothelium, megakaryocytic, and subendothelial tissues [1]. Each monomer of VWF is composed of identical repeated domains designated as A, B, C, D, and E [2,3]. The arrangement of the domains appears like a mosaic protein [4]. The basic features of each domain are given in detail (Table 1). The multimeric size of VWF is important for its function [5]. VWF in the circulating blood is found as a series of multimers ranging in size from about 500 to 20 000 kDa, the larger ones being hemostatically more effective [6].

An increase in VWF concentration in the blood is thought to be an indicator of endothelial injury [7,8]. VWF facilitates hemostasis via two separate pathways by stabilizing coagulation factor VIII and recruiting platelets to injured vessel wall or thrombi through the interaction with GPIb-α [9]. At the site of a wound or injury VWF helps platelets to adhere together to the walls of blood vessels and eventually lead to the healing process [10]. VWF carries another blood clotting protein called coagulation factor VIII to the site of clot formation [11]. This coagulation factor helps in platelet adhesion. At high shear rates the binding of VWF to collagen types I and III intercede platelet adhesion [12].

Recently, the crystal structure of A2 domain has been determined, and it shows high structural similarity with A1 and A3 domains and reflect that it adapts to shear sensor function [13]. Many reviews on VWF are available [14–16]. In this review our aim is to collect the recent information on VWF with special attention to A1, A2 and A3 subdomains for structure, function, metabolic network, and possible role in various diseases.

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Biosynthesis of von Willebrand factor

The biosynthesis of VWF is completed through a complex process, including several posttranslational modifications [17,18]. A 350-kDa polypeptide encoded by 9 kb VWF mRNA contains 2050 amino acid residue long mature polypeptide, 22 amino acids long signal peptide and propeptide of 741 amino acid residues. This pro-VWF rapidly dimerizes in the endoplasmic reticulum by disulfide formation [16,19]. VWF is synthesized in the endothelial cells and megakaryocytes [18]. The precursor undergoes multimerization after processing in the endoplasmic reticulum and the Golgi apparatus of the endothelial cell and is cleaved into two components; one is the mature protein, whereas the other is a propeptide of 97 kDa [20]. This mature protein mediates the interaction between platelets and components of the subendothelium at sites of vascular injury, and as a result these interactions lead to protection of the coagulation factor VIII from the proteolytic degradation. VWF is initially synthesized as a prepro-polypeptide. The pro-polypeptide can be cleaved and also be secreted. Free pro-polypeptide is identified as von Willebrand antigen II, a plasma glycoprotein of unknown function [10]. Plasma VWF consists of a heterogeneous series of multimers that are composed of an apparently single-type glycoprotein subunit, linked together by disulfide bonds [16,21].

Yamamoto et al.[22] observed that VWF is frequently used as a universal marker for endothelial cells and found that different levels of VWF antigen were detected in veins and arteries in mice. mRNA of VWF is present at elevated levels in lungs, spleen, aorta, and brain, and at low levels in liver, gut, skeletal muscle, and kidney [23]. In animals such as mouse, pig, the VWF expression is heterogeneous and varying in size, location, and type of vessel [24]. The liver is an important site of factor VIII synthesis. Factor VIII circulates in complex with VWF, and this interaction appears to prevent premature proteolytic cleavage and clearance of factor VIII [14,25]. High expression of VWF in the lung could promote homeostatic process that would help to protect against bleeding after injury through adhesion of cells at the alveolar capillary level in the lung [26].

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Secretion of von Willebrand factor

VWF is secreted as large multimers that are cleaved in the A2 domain by the metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13 (ADAMTS13), to give smaller multimers [27]. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower physiological shear stresses and causes thrombosis. VWF is secreted from endothelial cells through both the luminal and abluminal membranes [15]. Interestingly, through abluminal secretion some VWF is deposited into the vascular subendothelium, where it acts as an extracellular matrix protein to bridge circulating platelets [16]. Secretion of VWF from specialized storage granules of the endothelial cell, called Weibel–Palade bodies, is triggered by several substances [28]. These substances include some of important mediators of thrombosis and inflammation. Moreover, the regulated secretion of mature VWF also causes the release of equimolar amounts of the propeptide [29].

The secretion of VWF is stimulated by a variety of agents such as histamine, thrombin, fibrin β-adregenic agnoists, calcium ionophore A23187, and phorbol myristate acetate [30–32]. However, the magnitude of acute secretory response to epinephrine depends on the intracellular cyclic AMP levels [33]. It has been observed that desmopressin, a vasopressin analogue, induces a rapid, two-fold to three-fold increase in plasma VWF secretion [34]. Desmopressin itself is inactive on endothelial cells in vitro, and it is thought to produce an undefined secondary messenger [35]. The secretion of VWF occurs through two distinct pathways: one is continuous, which occurs without any cellular stimulation, whereas the other is regulated by storage granules [36]. On the contrary, a third pathway has also been described known as the basal secretion [37].

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Gene structure

VWF gene is located at the tip of the short arm of chromosome 12 [38]. Mancuso et al.[39] determined the gene structure of VWF and described the importance of various exons. VWF spans approximately 180 kb and contains 52 exons. The length of exons and introns vary from 40 to 1379 base pairs (bp) and 0.97 to 19.9 kb, respectively [39]. Furthermore, they have identified a number of repeats such as 14 Alu and an approximately 670 bp TCTA repeat in intron 40. Moreover, the 5′ flanking region of VWF contains an AT-rich region resembling TATA element and a repetitive sequence (GT)19[40]. The positions of intron–exon boundaries within the repeated amino acid sequence domains of VWF reflect that the evolution of these domains presumably occurs through the gene duplication [15]. The primary translation product predicted from the cloned cDNA of VWF shows a relationship between exons and various domains of VWF-polypeptide, and each relationship is well described earlier [41]. It may be suggested that each domain is encoded by one or many exons.

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Primary structure

Over 90% of the VWF precursors consists of four distinct repeated domains present in two to five tandem copies each in the order D1-D2-D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2 (Table 1) [39,42]. There are five types of repeated domains that approximately constitute 77% of the sequences. Domain A comprises approximately 180 amino acid residues and is present in three tandem copies between 1277 and 1871. The A domain of VWF is homologous to segments of complement factor B, complement factor C2, cartilage matrix protein, and α1-procollagen type I binding sites. The c domain of VWF is homologous to segments of thrombospondin and α 11-procollagen type I and type III [16]. The gene structure of VWF domain A3 and that of cartilage matrix show considerable variation in the placement of intron–exon boundaries with the exception at the 3′ ends of VWF domain A3 and both cartilage matrix protein [43].

The primary translational product consists of 2813 amino acids including a signal peptide [15]. Among the 2813 amino acids, 234 amino acids are cysteine, which accounts for exactly 8.3% content of cysteine. Cysteine is abundant in all domains except the triplicated A domain, which together contain only six cysteine residues. Interestingly, all the cysteine residues are conserved in mammalian VWFs. The mature subunit of VWF is glycosylated with 12 N-linked and 10 O-linked oligosaccharides, and the propeptide has three potential N-glycosylation sites [44]. Similar to the cysteine, the glycosylation sites are also conserved in all mammalian VWFs. At Asn384 and Asn468 of the mature subunit one or both of the oligosaccharides are sulfated [45]. Pro-VWF subunits dimerize through disulfide bonds in the neighborhood of the carboxy terminal following translocation into the endoplasmic reticulum. This tail-to-tail dimerization requires sequences of the last 150 residues. Both glycosylation and dimerization occur following the exit of the subunits from the endoplasmic reticulum [46].

The multimers of VWF in normal plasma range from 500 to 20 000 kDa that usually differ by the number of constituent VWF units. The VWF propeptide plays an important role in the assembly of multimers [47]. The D1 and D2 domains of the VWF propeptide contain CXXC sequence (Cys159-Gly-Leu-Cys162 and Cys521-Gly-Leu-Cys524), which resembles the functional site of thiol disulfide [48]. Insertion of an extra glycine into any of the sequences was compatible with the dimer formation in the endoplasmic reticulum, but the dimers were transported to the Golgi and secreted without forming multimers [49]. There are 23 cysteine residues present in between Lys273 and Lys448, out of which only six form intra-subunit disulfide bonds [50]. Multimer formation of VWF is mediated by a noncovalent interaction between two pro-polypeptides. In order to determine the involvement of different domains of VWF in the assembly to multimers, Voorberg et al.[51] have constructed and expressed a set of deletion mutants of full-length cDNA. They observed that both the D′ and the D3 domain are required for multimer assembly of VWF, including the pro-polypeptide chain. It has been demonstrated that deletion of these propeptide region of VWF resulted in the expression of only the dimeric form of VWF and prevented the synthesis of VWF multimers. Furthermore, the Tyr87Ser mutation in the pro-VWF region directly affects the multimerization and VWF biosynthesis [47]. Moreover, several naturally occurring mutations in the propeptide region of VWF (Tyr87Ser, Arg273Trp, Asn528Ser, Gly550Arg, Cys623Trp, and insertion of Gly625) showed an altered multimerization [47,52].

The multimeric size of VWF is regulated by the plasma metalloproteinase, ADAMTS13, which cleaves at a single site (Tyr1605-Met1606) in the A2 domain of VWF [53]. Recently, it was observed that residues from 1874 to 2813 of VWF (includes the VWF D4 domain) show excellent binding affinity [dissociation constant (Kd 86 nmol/l)] to ADAMTS13 [54]. It may be suggested that this novel D4-domain binding may be an initial step of a multistep interaction require for the proteolysis of VWF by ADAMTS13.

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Three-dimensional structure

The crystal structure of each subdomain of A domain has been determined, showing close structural homology. Here we are discussing the structural features of each domain separately in detail.

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Domain A1

The crystal structure of A1 domain has been determined in the native form [55] as well as a complex with NMC-4 fab [56,57], GPIb [58], GPIbα[59], and snake venom bitiscetin [60]. The overall fold of A1 domain is quite similar to that of A3 domain of VWF and the integrin I-domain (Fig. 1a). The structure represents a central hydrophobic parallel β-sheet flanked on two sides by amphipathic helices. A well ordered disulfide bridge is formed between Cys509 and Cys695, which connects the N-proximal and C-proximal sequences. The overall shape is cuboid, with six fairly flat faces [55]. On comparison with integrin I domains, this upper face contains the metal ion-dependent adhesion site (MIDAS) motif consisting of three closely spaced loops that together coordinate a magnesium ion [61].

Figure. No caption a...

The VWF-A1 contains a large number of charged amino acids, which form a salt-bridge network that wraps around the lower rim of the domain [62]. These stabilizing salt bridges are explained by the sensitivity of this region upon alanine mutagenesis [63]. There are four buried acidic residues: Glu557, which forms stabilizing salt bridges with two histidine side chains (His559 and His563); Glu626, which is buried but sits at the N-terminal end of helix α4 where it may be stabilized by the helix dipole; Asp514, which is buried and is stabilized by salt bridges formed by two arginine side chains (Arg552 and Arg611) forming a part of the salt-bridge network; and Asp520, which is buried below the upper face of the domain without a stabilizing salt-bridge partner [55,64,65].

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Domain A2

The crystal structure of A2 domain has been determined very recently [13]. It is unique among VWF-A domains. The structure of A2 is devoid of α4 helix; instead, it contains a loop and cis-proline (at 1645th position of mature polypeptide). This Cis-Pro of A2 domain plays a significant role in A2 domain refolding in the extracellular environment. Interestingly, this proline is also present in trans configuration at the corresponding position of A1 and A3 subdomains. Moreover, cistrans isomerization occurs in the folded and unfolded states, corresponding to formation and breakage of hydrogen bond with Arg1618, respectively [13]. The structural analysis of A2 domain reveals that it has two independent molecules [66]. It clearly represents hydrophobic β sheet core containing antiparallel β strands in the order β3, β2, β1, β4, β5, and β6 (Fig. 1b). Asp1806 of the A3 domain has a capping function similar to Asp1614 of A2 [13]. The dipoles of the α3- and α5-helices interact with charged, capping residues, Asp1614 and Arg1618, respectively, which are invariant in A2 domain [13]. The α6-helix ends in 2 Cys residues that are linked by an unusual vicinal disulfide bond that is buried in a hydrophobic pocket [62]. These features may narrow the force range over which unfolding occurs and may also show refolding. The A2 domain provides surprising insights into the structural specializations of a domain that has evolved to be a force sensor. The A2 domain is the only domain within VWF that is not protected from unfolding by medium-range or long-range disulfide bonds.

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Domain A3

Three-dimensional structure of A3 domain has been reported by many groups independently [61,67–69]. The VWF A3 domain adopts a dinucleotide-binding fold [67], consisting of a central β sheet with six strands that is surrounded by seven α helices. All β strands are parallel, except for strand β3, which is located at an edge of the sheet (Fig. 1c). The disulfide bridge between residues Cys923 and Cys1109 connects the N-terminal and C-terminal regions, and denotes the domain boundaries. The interesting feature of A3 domain is the collagen-binding site showing a markedly high concentration of negatively charged residues. This region encompasses a potential metal-binding site containing the motif DXSXS, which is required for ligand interaction in the homologous I-type domains of integrins CR3 and LFA-1. The structure of the A3 domain suggests that adhesion to collagen is primarily achieved through interactions between negatively charged residues on A3 and positively charged residues on collagen [69]. The absence of a pronounced binding groove precludes a large van der Waals surface interaction between A3 and collagen and is consistent with the low affinity for collagen of a single A3 domain and the requirement for multimeric VWF for tight association with collagen [70].

The main chain atoms of the central β-sheet overlap with the integrin αMI domain with an rms deviation of 0.5 Å. There are several surprising features of the structure: the α2-helix is replaced by a loop and helix α3 is extended by five residues; at the top of the domain, the βE-α6 loop adopts a different conformation; and α7-helix is only two turns long and is followed by an abrupt 90° turn before forming a new 8th helix ending with the C-terminal Cys-1109. The solvent accessible surface of the A3 domain is predominantly hydrophilic with the exception of a region located at the bottom and back face of the molecule [67]. Residues contributing to this hydrophobic region are Val984, Val985, Pro986, Leu994, and Val997. The proximity of this hydrophobic region to the N-terminal and C-terminal residues is consistent with a potential hydrophobic interdomain interaction site.

The structural comparison among A1, A2, and A3 domains reveals that they all contain similar fold (Fig. 1d). The rms deviation of backbone carbon atoms of A1 domain with that of A2 and A3 is 1.91 and 2.746 Å2, respectively, while the rms deviation of A2 domain with A3 is only 1.597 Å2, which reveals that A2 and A3 are more similar structurally as compared to A1 domain.

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Biological functions

VWF performs a large number of functions in the human body [14–16,71]; binding to factor VIII and platelet surface glycoprotein is prominent among them [6,72,73]. The binding of VWF to platelets is regulated by its initial interaction with connective tissue and also by shear stress in flowing blood. We have predicted the functional protein–protein interaction network using an online server STRING [74]. VWF shows interaction with various proteins in order to perform many important functions (Fig. 2 and Table 2). The biological functions of VWF are described here in detail.

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Platelet adhesion

VWF does not interact with circulating platelets in the absence of injury [75]. However, in the course of damage the endothelium allows VWF to bind with constituents of subendothelial connective tissue. Another report suggests that platelet and collagen adhesion increases the platelet aggregation induced by binding of VWF [76]. Site-directed mutagenesis studies on A2-domain showed that Arg834Gln and Arg834Trp mutant showed a remarkable decrease in platelet adhesion to collagen type III under flow conditions [77]. The Arg-Gly-Asp sequence (1744–1746 of mature polypeptide) of VWF is considered as a recognition site for integrin receptor, which promotes endothelial cell adhesion [78].

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Binding to collagens

VWF binds specifically to several collagens in vitro including types I, II, III, IV, V, and VI, but it does not bind to the denatured gelatin [69]. Platelet adhesion is also supported by fibrillar collagen of type I or type III under high shear condition [79]. Binding sites for fibrillar collagen have been found in the A1 and A3 domains of VWF [67,80]. But the major collagen-binding site of multimeric VWF is found in the domain A3.

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Binding to platelet GPIb

The VWF has strong affinity for platelet GPIb [81]. Such kind of interaction requires the glycoprotein (GPlb IX-V complex), with significant affinity for VWF. GPIb serves as one of the essential receptors for VWF and plays a crucial role in platelet thrombus formation [82]. Undeniably, the binding of multimeric VWF to GPIb results in platelet activation and may lead to adhesion and aggregation. GPlb-IX-V consists of at least four polypeptide chains encoded by separate genes: GPIbα, GPIbβ, GPIx, GPv. GPIbα, and GPIbβ are disulfide-linked and associate noncovalently with GPIbα chain [83,84]. Optimal binding requires sulfation of tyrosine residues at positions 276, 278, and 279 [85]. VWF-dependent platelet adhesion occurs optimally under conditions of high fluid shear stress [82]. The small bacterial glycopeptide antibiotic ristocetin dimerizes and binds to both VWF and platelets and causes platelet aggregation by inducing VWF to bind GPIbα[86]. The GPIb-binding site on VWF is localized in A1 domain [84]. VWF in solution does not bind tightly to platelets because the intrinsic affinity of each binding site is low. Multivalent binding is difficult in solution because of the conformational flexibility of VWF [87]. Immobilization of VWF in connective tissue could restrict the motion of multiple binding sites, distributing them favorably to bind several GPIb receptors and retain platelets at the vessel wall [88].

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Binding of von Willebrand factor to platelet integrin αIIββ3

The αIIββ3 is a member of the integrin family of cell-surface receptors that does not bind to VWF. When platelets are activated by thrombin, αIIββ3 is able to bind fibrinogen, fibronectin, or VWF with high affinity [89]. This interaction is not sufficient for platelet adhesion, but specific inhibition of αIIββ3 function impairs the adhesion of platelets to surfaces coated with either VWF or collagen. Therefore, αIIββ3 may contribute to platelet adhesion that is initiated through GPIb binding to VWF [90,91].

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Fluid shear stress

The VWF-mediated platelet adhesion depends on fluid shear stress [11]. As blood flows through the vessel, velocity is maximum at the center and falls to zero at the vessel walls [86]. In the formation of a thrombus, platelets must bind to the vessel wall despite these opposing forces. It has been observed that at low shear rates of veins and normal arteries, platelet adhesion is not stimulated by VWF. However, at shear rates greater than 1000 s−1, platelet adhesion is strongly dependent on VWF [92]. The rapid interaction between GPIbα and VWF promotes the reversible tethering of platelets that approach the wall [93]. The slowly moving tethered platelets adhere irreversibly through the slow binding of cell surface αIIβ3 to ligands in connective tissue [94]. Keuren et al.[95] showed that the C-domain of VWF is a critical determinant of platelet adhesion to fibrin under conditions of high shear stress. Moreover, the transition from GPIb-dependent to integrin-dependent binding is accelerated by platelet activation, which can be mediated by constituents of connective tissue such as collagen or by soluble platelet agonists including ADP or epinephrine [96].

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Stabilization of blood coagulation factor VIII

Patients with hemophilia have low levels of factor VIII and normal levels of VWF, whereas patients with severe von Willebrand disease (VWD) have undetectable levels of VWF and factor VIII. This behavior reflects the dependence of normal factor VIII survival on the formation of noncovalent interaction between factor VIII and VWF [11]. It has been observed that all sizes of VWF multimers bind to factor VIII with similar affinity [85]. In fact, almost all VWF subunits can bind with factor VIII, but adsorption of VWF to surfaces markedly decreases the stoichiometry of binding. The factor VIII-binding site on VWF is located within the amino terminal 272 amino acid residues of the mature subunit [97]. The corresponding VWF-binding site on factor VIII is near the amino terminus of the 80 kDa light chain. The factor VIII light chain consists of residues 1649–2332 of the factor VIII precursor. The VWF-binding site involves amino acids in the segment between residues 1669–1689, and optimal binding requires sulfation of Tyr1680 [85]. When factor VIII is activated during blood coagulation, thrombin cleaves it at Arg1689. This cleavage destroys the VWF-binding site and releases factor VIIIA [98]. In type IIN VWD a mutation in D′ domain of VWF (Gln290His and Cys297Arg in mature VWF sequence) causes a dramatic decrease in the binding of VWF to factor VIII [99].

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Binding to sulfatides

Sulfatides are sulfated glycosphingolipids present on cell surfaces that bind to adhesive proteins including VWF. VWF binds specifically with high affinity to sulfated glycolipids. Such binding probably provides VWF the ability to agglutinate erythrocytes as evident from two other sulfatide-binding proteins, such as laminin and thrombospondin [100]. Interestingly, characteristics of VWF binding to sulfatides resemble those of VWF binding to platelets. Studies have showed that the sulfatide-binding site of VWF is located in amino acid residues Gln626–Val646 in the A1domain. Interestingly, the A1 domain also contains the binding site for GPIb, a site that has been reported to be distinct from the sulfatide-binding site. Moreover, the interaction of sulfatides with VWF affects GPIb-mediated platelet adhesion under flow conditions [101]. Another finding by Christophe et al.[102] suggests that the sequence 569–584 of VWF is a part of the interactive site for sulfatides or may influence its activity that is distinct from the domain involved in heparin binding. Nakayama et al.[103] performed alanine mutation, and they observed that 10 mutants showed decreased sulfatide-binding including four novel mutations at Arg1392, Arg1395, Arg1399, and Lys1423.

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Binding to heparin

It has been demonstrated that there is significant impairment of VWF function in the plasma of acutely heparinized patients. VWF is also known to bind heparin through the domain that lies in close proximity to GPIb binding domain in the region amino acids 449–728 [104]. Heparin can inhibit VWF platelet hemostatic interactions. It has been suggested that heparin may interfere with platelet function, and therapeutic doses of heparin can prolong the bleeding time. Furthermore, heparin could inhibit ristocetin-mediated platelet agglutination. All these activities are associated with heparin due to its strong affinity for VWF [105]. Using synthetic peptide binding studies it was suggested that the 23-residue sequence (Tyr565-Ala587) of VWF retains the consensus motif that binds heparin with appreciable affinity [105]. Sobel et al.[104] investigated the mechanism of heparin-induced inhibition of VWF function and suggested that the effect of heparin is independent of ristocetin, and heparin most likely acts not at the platelet surface but in solution by binding VWF and impairs the capacity of VWF to bind the platelet.

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Von Willebrand disease

Von Willebrand disease (VWD) is an inherited genetic bleeding disorder that can be inherited from either parent and affects males and females equally [106,107]. VWD is a congenital extrinsic platelet defect resulting in platelet dysfunction [108] and is commonly reported in dogs, swine, horses, cattle, and cats. This autosomally inherited disorder is caused by either qualitative (type II) or quantitative (types I and III) deficiency of VWF. VWD results primarily in abnormal primary hemostasis (platelet plug formation) and prolongation of bleeding time in vivo caused by a lack of functional VWF [109].

The release of VWF is enhanced by involvement of some modulators such as histamine, fibrin, and estrogen [110]. Furthermore, the release of α granules of platelets is stimulated by collagen, platelet-activating factor, thrombin, and adenosine diphosphate [110]. Although there are small, medium, and large multimers of VWF, the multimers with high molecular mass are most active during hemostasis because they have large numbers of binding sites per molecule and their physical characteristics can be altered under certain conditions of blood flow [111]. Broadly, there are four hereditary types of VWD: type I, type II, type III, and platelet-type [112].

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Type I

Type I is the most common and mildest form of VWD [113]. The main symptom of type I VWD is a significant mucocutaneous bleeding history. In type I, the level of VWF in the blood and the levels of factor VIII may also be reduced [114]. The condition becomes worse by taking aspirin and other nonsteroidal anti-inflammatory drugs. People with type I VWD usually do not bleed spontaneously but can have significant bleeding with trauma, surgery, or tooth extraction [115]. Recently, Othman et al.[116] have studied the effect of a 13-bp deletion in the promoter of the VWF gene in a patient with type I VWD. They showed that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type I VWD.

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Type II

Type II is accounts for 15–20% of VWD cases [117]. It is a variant of the disease with primarily qualitative defects of VWF (abnormal function) [118]. This disease can be either autosomal dominant or autosomal recessive. The type II VWD can further be classified into four subtypes: IIA, IIB, IIM, and IIN. The type IIA VWD is by far the most common [119]. Bernardi et al.[120] detected two transversions in two unrelated type IIA VWD: a C to A at nucleotide 8821 and a T to A at nucleotide 8830, resulting in the missense mutations Pro864His and Val867Glu, respectively.

Type IIB VWD is a rare autosomally inherited bleeding disorder, usually characterized by enhanced ristocetin-induced platelet aggregation in platelet-rich plasma [121]. Type IIB VWD has typically characteristic features: increased ristocetin-induced platelet agglutination at low ristocetin concentration, selective loss of plasma high-molecular-weight multimers, and mutations involving exon 28 of the VWF gene that codes for the A1 domain of VWF, the known contact site for platelet GPIb [118,122]. This is a disease with an autosomal dominant inheritance, characterized by an abnormal VWF structure resulting in enhanced affinity to platelet glycoprotein GPIbα that may result in moderate-to-severe thrombocytopenia [123].

Type IIM is also a type II VWD, characterized by a decreased platelet-directed function that is not due to a decrease in high-molecular-weight multimers [121]. It also occurs due to the decreased VWF activity [124]. The mutations Glu561Ser, Phe606Ile, and Ile662Phe, observed in VWD type IIM, have decreased VWF:RCo and retain normal botrocetin-induced binding to platelet GPIb. In addition, charged-to-alanine mutations at Glu626, Asp520, Lys534, Lys48, Lys534, Lys569, Lys642, Lys64, and Lys549 of VWF resulted in reduced ristocetin-induced binding, but not botrocetin-induced binding of VWF to platelets [125].

The type IIN VWF is also rare and is characterized by a decreased affinity for factor VIII. In type IIN VWD, the presence of abnormal VWF with an altered factor VIII-binding site results in diminished or no factor VIII–VWF interaction, which leads to a defective coagulation with generally normal primary hemostasis. It is recessively inherited disease and clinically similar to mild hemophilia. Till now there are seven of the eight reported missense mutations associated with type IIN VWD located in the D′ domain. A mutation Asp879Asn in prepro-VWF (position 116 in mature VWF) resulted in decreased factor VIII due to an induced conformational change in the D′ domain [126]. Another missense mutation (Thr791Met) is shown to induce a factor VIII binding defect. A large number of mutations have been reported in type IIN VWD by Mazurier et al.[127].

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Type III

Type III is an autosomal recessive inherited bleeding disorder and the most severe form of VWD due to being homozygous for the defective gene. Type III VWD is much less common with frequency estimated at approximately one per million. This disease results in a remarkable decrease or absence of VWF and is associated with moderate-to-severe bleeding symptoms (epistaxis, menorrhagia, arthropathy, and postoperative bleeding) [128]. Major clinical features involve excessive mucocutaneous bleeding and prolonged oozing after surgical procedures. Patients with type III VWD may have severe bleeding not only in the mucocutaneous areas but also in the joints because factor VIII is also very reduced. Furthermore, hematomas and hemarthrosis are also observed because factor VIII levels are as low as in moderate–mild hemophiliacs [129]. There are a large number of mutations reported in type III VWD. Interestingly, most mutations identified are ‘null’ mutations, resulting in a lack of expression of VWF due to nonsense, frame shift, splice site, and large deletion mutations.

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Platelet-type von Willebrand disease

Platelet-type VWD (PT-VWD), also known as pseudo-VWD, is an autosomal dominant type of VWD caused by mutations of the VWF receptor on platelets, specifically the GPIbα[130]. PT-VWD is usually misdiagnosed and considered as type IIB VWD, which shows similar bleeding and phenotypic profiles [131]. This disease results from an abnormally high affinity interaction between the platelet membrane glycoprotein Ib/IX/V complex and VWF. Othman et al.[132] reported three missense mutations for PT-VWD, Gly233Val, Met239Val, and Gly233Ser, which are located within the C-terminal disulfide loop of the 45-kDa domain of GP1bα, and a 27-bp deletion in the macroglycopeptide-coding region of GP1bα. Such mutations cause structural changes in GP1bα, and are responsible for the PT-VWD phenotype [133].

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Clinical measurements of von Willebrand factor

The assay of VWF is necessary for diagnosis of the quantitative VWD. Numerous methods are available for the clinical quantification of VWF in blood plasma [134]. VWF activity may be assayed by measuring VWF antigen, or the ratio of VWF:ristocetin cofactor (RCo) or the ratio of VWF:collagen binding activities. The most used method for VWF:RCo assay depends on ristocetin-induced platelet aggregation or agglutination. Recently, a simple method is developed for VWF measurement that is based on flow cytometry having superior accuracy, specificity, and sensitivity [135]. The VWF and collagen binding (VWF:CB assay) can significantly reduce the diagnostic error rate as compared to the VWF:RCo assay [136–138]. Later, VWF-FVIII binding assay has been used for the investigation of patients with suspected VWDIIN [139]. Interestingly, such an assay provides an accurate measurement of VWF:FVIII binding activity and helps in the identification of homozygous patients with VWDIIN and heterozygous carriers. Day by day various new methods are emerging for the accurate diagnosis of VWD [140,141].

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It is evident from the analysis of VWF that it is a clinically important protein. Therefore, we extensively studied VWF in terms of its structure and functions. VWF is synthesized exclusively in endothelial cells and megakaryocytes. It shows a strong binding with collagen, integrin GPIb, factor VIII, platelet integrin αɪɪββ3, and several other compounds. VWD is an inheritable bleeding disorder that affects the clotting ability of blood. This disease may further be classified into type I, type II, and type III diseases. The structure analysis of VWF is critically important for the development of structure and function relationship and its association with diseases.

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M.I.H. is grateful to the Department of Science and Technology, Government of India for the award of young scientist fellowship. F.A. is especially grateful to the Council of Scientific and Industrial Research, and University Grants Commission, Government of India, for financial assistance.

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Conflict of interests

The authors state they have no conflicts of interest.

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biomarker; blood clotting; cell adhesion; fibrinogen-binding; hemostasis; integrin GPIb; von Willebrand diseases; von Willebrand factor

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