Ann Hematol (1996) 72 : 341–348
Q Springer-Verlag 1996
REVIEW ARTICLE
M. Furlan
Von Willebrand factor: molecular size and functional activity
Received: 7 March 1996 / Accepted: 13 March 1996
Abstract Von Willebrand factor (vWF) is the largest protein found in plasma. It circulates in blood as a series of multimers ranging in size from 500 to 20 000 kDa. The variable molecular weight of vWF is due to differences in the number of subunits comprising the protein. vWF mediates platelet adhesion to subendothelium of the damaged blood vessel. Only the largest multimers are hemostatically active. Each vWF subunit contains binding sites for collagen and for platelet glycoproteins GPIb and GPIIb/IIIa. Multiple interactions of repeating binding sites in vWF multimers with adhesive protein(s) of the subendothelium and with receptors on the platelet surface lead to “irreversible” binding of platelets to the exposed subendothelium. Functional properties of vWF are typical of multisubunit proteins encoded by autosomal loci. The phenotype of von Willebrand disease is determined by the properties of the dysfunctional subunits which become incorporated into heteropolymeric forms of vWF. Absence of large vWF multimers, seen in type 2A von Willebrand disease and in myeloproliferative disorders, is associated with bleeding tendency. On the other hand, in patients with vWF multimers of supranormal size, as they occur in thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), there is an increased risk of thrombosis. Proteolytic enzyme(s) are involved in physiologic regulation of the polymeric size of vWF. We have purified from human plasma a protease cleaving vWF at the same peptide bond that is also cleaved in vivo. vWF was quite resistant against the protease in a physiologic buffer but was degraded at low salt concentration or in the presence of 1 M urea. It appears that a conformational change in the vWF
Supported by grants from the Swiss National Science Foundation (Grant 3200-037435.93) and from the Central Laboratory, Blood Transfusion Service, Swiss Red Cross M. Furlan Central Hematology Laboratory, Inselspital, University of Bern, CH-3010 Bern, Switzerland
molecule exposes the specific protease-sensitive peptide bond and thus enhances degradation of vWF multimers. In some variants of type 2A vWF, the cleavage site in the vWF subunit is more susceptible to proteolytic degradation than in normal vWF, whereas in patients with TTP or HUS the protease activity may be suppressed. vWF-degrading protease plays an important role in pathogenesis of congenital or acquired disorders of hemostasis and thrombosis. Key words von Willebrand factor 7 von Willebrand disease 7 Binding affinity 7 Multimers 7 Phenotype
Introduction Von Willebrand factor (vWF) is a multimeric plasma glycoprotein with two distinct biological functions: it serves as the carrier for procoagulant factor VIII and protects it from inactivation by activated protein C and factor Xa in the circulating blood, and it mediates platelet adhesion to subendothelium of the damaged blood vessel. vWF and factor VIII circulate in blood plasma as a noncovalently associated complex consisting of about 99% vWF and 1% factor VIII. A decreased concentration in the level of vWF or an abnormality in the interaction between vWF and factor VIII cause a shortened half-life of factor VIII in the circulating blood and thus a decrease in the level of factor VIII activity. Binding of platelets to the exposed subendothelium is the initial step in the formation of a hemostatic plug. vWF deficiency or abnormality lead to von Willebrand disease (vWD), which is now known to be the most common inherited human bleeding disorder. Bleeding symptoms in patients with vWF are nose and gingival bleeding, bleeding from minor skin wounds and after tooth extraction, postoperative bleeding, and menorrhagia. vWF is synthesized exclusively by vascular endothelial cells and megakaryocytes. The vWF gene, consisting of about 180 kilobases and containing 52 exons, is located at the tip of the short arm of chro-
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mosome 12. Since the amino acid sequence of vWF was determined, one decade ago, a large number of different mutations have been discovered that cause vWD and provide important information about the relationship between structure and biological functions of vWF. General reviews on vWF structure and function [1, 2] and on the pathogenesis of vWD [3, 4] have been published elsewhere.
Structure and biological functions vWF multimers are composed of a 270-kD polypeptide subunit comprising 2050 amino acid residues. Each subunit contains binding sites for collagen [5–8] and for platelet glycoproteins GPIb [9–12] and GPIIb/IIIa. In intact blood vessels, vWF does not interact with the platelet receptors. It is assumed that in the injured blood vessel, the subendothelial structures become exposed and bind vWF; this interaction seems to induce a conformational change in vWF leading to exposure of binding sites for GPIb and to platelet adhesion. It has not yet been established with certainty which components of the subendothelial matrix interact with vWF in vivo. vWF binds not only to fibrillar collagen types I, III, and VI of the vessel wall, but also to noncollagenous components of the subendothelium [13]. Furthermore, binding of vWF to hydrophobic plastic [14] or glass [15] surfaces and high shear stress [16] were shown to “activate” the GPIb binding site in vWF. The interaction of vWF with the positively charged antibiotic ristocetin [17] or reduction of the negative charge by removal of sialic acid residues from the carbohydrate side-chains of vWF also result in exposure of the GPIbbinding site [18]. Botrocetin, a protein isolated from the venom of certain pit vipers, particularly Bothrops jararaca, was shown to bind to vWF in proximity to the GPIb binding site and to promote platelet agglutination in the presence of vWF [19, 20]. The direct binding of asialo-vWF to unstimulated platelets provides evidence that GPIb receptor is normally exposed on the membrane of the resting platelet. Binding of vWF to GPIb induces platelet activation, resulting in expression of the complex GPIIb/IIIa on the platelet membrane [21– 23]. “Activated” GPIIb/IIIa is capable of cross-linking adjoining platelets via bridges made by vWF, fibrin(ogen), fibronectin, vitronectin, and other proteins containing the Arg-Gly-Asp sequence.
Size and activity of vWF vWF circulates in plasma as a series of multimers containing a variable number of subunits. vWF is the largest known protein present in human plasma. Flexible strands of up to 2 mm [24] are comparable in length to the diameter of a medium platelet. The largest multimers may be as large as 20 000 kD. Other large plasma proteins with Mr`500 kD also consist of multiple poly-
peptide subunits or of repeating domains, e.g., haptoglobin 2-2 (Mr up to 1000 kD), IgM (Mrp971 kD), a2macroglobulin (Mrp725 kD), C4-binding protein (Mrp540 kD), and apolipoprotein B-100 (Mrp 514 kD), but their molecular size is more than one order of magnitude smaller than that of the largest vWF multimers. The multimeric pattern of vWF seen on sodium dodecyl sulphate (SDS)/agarose electrophoresis shows regularly spaced bands, suggesting that each multimer differs from the adjacent ones by a constant molecular mass of about 500 kD [25, 26]. Multimer formation takes place in the endoplasmic reticulum: initially, dimers are assembled from pairs of polypeptide subunits via disulfide bridges between cysteine residues located in the carboxy terminal regions; subsequently, multimers are formed by interdimeric disulfide linking of amino terminal domains [27]. Only the large multimeric forms of vWF are hemostatically active. In patients with bleeding disorders, both the amount and the quality of vWF have to be examined. The functional activity of vWF is measured by the conventional ristocetin assay, in which agglutination of formalin-fixed normal human platelets by vWF present in patient plasma is determined turbidimetrically. This platelet-agglutinating property of vWF is denoted as ristocetin cofactor activity. The size distribution of vWF is analyzed by agarose gel electrophoresis in the presence of SDS. Multimers can be visualized within the gel by radiolabeled antibodies, or they can be detected by immunoblotting techniques using enzyme-labeled anti-vWF antibodies. Single bands appear in 1% agarose gels (Fig. 1); each band can be further resolved into a number of satellite bands (triplets, quin-
Fig. 1 Subunit composition of von Willebrand factor. Electrophoresis of normal nonreduced plasma vWF in SDS-1% agarose gel (left panel) separates vWF multimers according to their size. The sample was applied on top of the gel. After electrophoresis, the proteins were electrotransferred to nitrocellulose and the bands of vWF were detected using peroxidase-labeled rabbit IgG against human vWF. The bottom band, having the most rapid anodic mobility, corresponds in size to the dimer (MWF500 kD) of the vWF polypeptide subunit (middle panel). Each slower migrating band is one dimer larger than the faster one. The arrow (right panel) schematically illustrates the decrease of the functional activity (binding affinity for collagen or platelet receptor GPIb) in small molecular forms of vWF
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tuplets) by SDS-electrophoresis in gels of higher agarose concentration (2–3% agarose gels). It has been shown by gel filtration of cryoprecipitate on large-pore agarose that the large multimers eluting in the void volume have a considerably higher ristocetin cofactor activity per unit antigen than the later-eluting vWF species of smaller size [28]. Gentle disulfide reduction of large vWF multimers resulted in decreasing molecular size and loss of ristocetin cofactor activity; small molecular forms of vWF were adsorbed onto colloidal gold granules and thereby increased their platelet-agglutinating activity [29]. vWF multimers of different sizes were fractionated by gel filtration and the resulting fractions subjected to binding studies in the presence of ristocetin (vWF binding to GPIb) or after addition of thrombin (vWF binding to the complex GPIIb/IIIa). These studies showed up to ten times higher Kd values for smaller molecular forms of vWF than for larger multimers [30]. Ristocetin-induced agglutination of washed platelets as well as thrombin-induced platelet aggregation in platelet-rich plasma were significantly faster with large multimers than with small molecular forms of vWF. These investigations further support the view that the affinities for platelet receptors (GPIb and GPIIb/IIIa) of vWF are related to its multimeric size.
Multivalent binding of polymeric vWF to platelets and collagen Adhesion of platelets from flowing blood to collagen may occur in the absence of vWF. Three candidate receptors for collagen have been postulated: GPIa-IIa (also called VLA-2), GPIV (denoted also as CD36), and GPVI. However, a decreased level or an abnormality of vWF results in reduced platelet adhesion to subendothelium under conditions of high shear stress and in prolonged bleeding time, indicating that direct interactions of exposed subendothelium with platelets via specific collagen receptors are insufficiently strong to resist remarkable shear forces encountered in the circulating blood, particularly in small vessels and stenosed arteries. The importance of the multimeric structure for function of vWF was confirmed by binding studies using vWF of different molecular sizes. A straight line was obtained in the Scatchard plot for interaction of human fibrillar collagen type III with the dimeric proteolytic vWF fragment SpIII consisting of two N-terminal polypetide remnants (residues 1–1365), and the calculated association constant Ka was found [6] to be in the range between 1.5 and 3.0!10 P6 M P1. Similar binding affinity (Kap0.5–1.0!10 P6 M P1) was reported for binding of the monomeric proteolytic fragment SpI (residues 911–1365) [7] or recombinant vWF-A3-domain (residues 908–1111) [8] to calf skin type I collagen. On the other hand, Scatchard plot for binding of polymeric vWF to collagen showed binding behavior typical of multiple classes of binding sites [6] with an apparent high affinity Ka of 1–3!10 P8 M P1. Also the
monomeric glycosylated recombinant vWF-A1-domain (residues 475–709) had about 100 times lower affinity for GPIb (Kap0.22!10 P6 M P1) [11] than the polymeric human vWF [30, 31]. These data show that the multimeric structure is not absolutely essential for interaction of vWF with collagen or with platelet receptors, but the presence of repeating subunits in each multimer may contribute to considerably stronger interactions required for stable adhesion of platelets to subendothelium in the flowing blood. A model of vWF binding to GPIb receptors situated on the platelet surface is depicted in Fig. 2. The starting state is shown on the left: multivalent ligand (vWF) in solution binds to receptors with forward and reverse rate constants k1 and kP1, respectively. The association constant is K1pk1/kP1 and the resulting ratio of the bound to free n1 K1 r p ligand concentration can be written as . F 1 1cK1 F Singly bound vWF multimer can bind to a second receptor with rate constant k2. The latter constant depends upon concentration of receptors at the proper distance, capable of establishing a bond with the adjacent vWF subunit. About 20 000–25 000 copies of GPIb are present on the surface of a human platelet [32]. Thus, the platelet surface is only sparsely covered with GPIb receptors that appear to be randomly dispersed in the platelet membrane and do not move during platelet activation [33]. Since the second equilibrium constant K2 depends on appropriate spacing of platelet surface receptors, it will presumably be significantly smaller than K1. Theoretically, the bound/free fraction n2 K 1 K 2 r p of the doubly bound ligand is . F 2 1cK1 K2 F Binding of the third functional site to the third receptor will proceed with a rate constant similar to that for binding of the singly bound vWF to the second recepn3 K1 K 22 r ; . Finaltor, resulting in K3;K2 and F 3 1cK1 K 22 F
12
12
12
Fig. 2 Binding of multivalent vWF to GPIb receptors on platelet surface. A highly simplified sequence of binding steps is shown, assuming appropriate spacing of receptors with regard to the separation distance between proximal functional groups. Singly bound ligand can either dissociate with rate-constant kP1 or bind to a second receptor with rate-constant k2. Doubly bound ligand can return to its singly bound state or become triply bound. Equilibrium constants for these interactions K1`K2FK3 are postulated
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ly, in a mixture of vWF multimers of variable size (composed of up to i dimers), the fraction of bound vWF i ni K1 K iP1 r 2 will be approximately ; A . Even if F ip1 1cK1 K iP1 F 2 the contribution of each subsequent equilibrium constant K2 is small in comparison to the initial association constant K1, additional bonds established by multiple interactions of repeating binding sites with surface receptors may dramatically increase the stability of the complex. Sufficient number of bonds represents the “irreversible” binding between vWF multimers and platelets.
Proteolytic degradation of vWF The largest multimers are present in storage granules both in platelets (a-granules) and in endothelial cells (Weibel-Palade bodies), from which they are released into circulation [34]. Lower molecular weight forms of vWF in circulating blood were found to contain increased amounts of proteolytically cleaved fragments [35]. It has been shown that the satellite bands represent proteolytic degradation products of vWF in which terminal dimeric subunits have been cleaved by a proteolytic enzyme [36, 37]. It has been suggested, on the basis of the N-terminal amino acid sequence of the circulating degradation fragments of vWF, that the peptide bond Tyr842–Met843 is cleaved by a calpain-like protease [38]. It appears that the size distribution of vWF multimers may be regulated by proteolytic degradation of circulating vWF. If the proteolytic degradation of vWF is too fast, as in some congenital variants of vWF [39, 40] or in some myeloproliferative disorders [41], the consequence is defective hemostasis. On the other hand, in patients with extremely large vWF multimers, as they occur in thrombotic thrombocytopenic purpura (TTP) [42], there is an increased thrombotic tendency. It remains to be investigated whether the pathogenesis of the latter disorder is associated with a deficiency or abnormality of a protease capable of degrading very large vWF multimers to smaller molecular forms. Our preliminary experiments have shown an impaired activity of the vWF-degrading protease in some patients suffering from TTP or HUS (unpublished observation). We have isolated from human plasma a vWF-cleaving protease using affinity chromatography and gel filtration [43]. An estimated purification factor of about 10 000 has been achieved. The proteolytic activity was found to be associated with a protein of MrF300 kD. High-molecular-weight vWF was apparently resistant against degradation when incubated with the purified enzyme preparation in a physiologic buffer, but it became degraded at low salt concentration or in the presence of 1 M urea (Fig. 3). It appears that low ionic strength or urea affect the conformation of the vWF molecule and thus expose the cleavage site. No degradation of three other proteins (human fibrinogen, bovine
Fig. 3 Influence of low ionic strength and urea on proteolytic degradation of von Willebrand factor. A purified preparation of vWF was incubated with purified protease in a physiologic buffer (0.15 M NaCl), in 5 mM Tris buffer (in the absence of NaCl), at physiologic ionic strength in the presence of urea (0.15 M NaClP1 M urea) and at low ionic strength in the presence of urea (5 mM TrisP1 M urea). Digested material was submitted to SDSelectrophoresis in agarose gel and vWF was detected by immunoblotting using peroxidase-labeled IgG against human vWF
serum albumin, and calf skin collagen) by the purified protease was observed under the same experimental conditions. This suggests that the protease possesses high specificity for vWF. Proteolytic activity had a pH optimum at 8–9 and was not affected by serine enzyme inhibitors or sulfyhdryl reagents. Inhibition by chelating agents was best reversed by barium ions. The observed properties of the vWF-degrading enzyme differ from those of all hitherto described proteases. Analysis of degraded vWF showed that the peptide bond Tyr842– Met843 had been cleaved – the same bond that had been proposed to be cleaved in vivo [38].
Pathogenesis and classification of von Willebrand disease vWD, the most common congenital bleeding disorder in humans, with a prevalence of about 1%, is the consequence of quantitative and/or qualitative defects of vWF. All vWD is caused by mutations at the vWF locus. Type 1 vWD refers to partial quantitative deficiency of vWF and is the most frequent form of the disease, accounting for about 70% of all cases. Type 2 vWD refers to qualitative deficiency of vWF. Type 3 vWD is characterized by the virtual absence of detectable vWF in plasma and platelets and has a prevalence of approximately 1 in 1 000 000 subjects.
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Type 2 vWD comprises four different subtypes and is phenotypically very heterogeneous. Common to all subtypes of type 2 vWD is the occurrence of qualitative abnormalities of vWF resulting, in most cases, in abnormal multimeric structure of the molecule. vWD variants with decreased platelet-dependent function, which is attributed to the lack of hemostatically effective large vWF multimers, are classified under type 2A. In the laboratory, these variants are recognized by a disproportionately low value of functional vWF level compared with the values of the vWF antigen. Most type 2A vWD is due either to impaired secretion of large multimeric forms of vWF or to enhanced proteolytic degradation of the large vWF multimers. To investigate the molecular mechanism responsible for the absence of large multimers in type 2A vWD, processing of recombinant vWF with different known type 2A mutations has been investigated. Impaired secretion of high-molecularweight multimers was observed in variants Ser850 ] Pro [44], Gly742 ] Arg, Ser743 ] Leu, Val844 ] Asp [45], and Leu777 ] Pro [46], whereas secretion of large multimers similar to wild-type vWF was noted with Gly742 ] Glu, Arg834 ] Trp [45], and Ile865 ] Thr [46]. The above mutations are located within the stretch of amino acid residues 742–865. Apparently, all of these mutations perturb a sensitive structure in the vWF molecule, and the resulting structural alterations interfere with the intracellular polymerization and/or transport, or they result in increased susceptibility of vWF to proteolytic degradation at the peptide bond Tyr842–Met843. Recombinant type 2A mutants Arg834 ] Trp and Arg834 ] Gln were rapidly degraded by the purified vWF-degrading protease in a buffer of physiologic ionic strength in the absence of urea, while the wild-type vWF was hardly affected under the same experimental conditions (data not shown). These results confirm that mutations in the A2 domain may lead to an enhanced proteolytic sensitivity of vWF. The absence of high-molecular-weight multimers is usually observed also in type 2B vWD, but this deficiency is caused by increased binding affinity of variant vWF for platelet receptors GPIb, resulting in an accelerated clearance of the most adhesive, largest vWF multimers from plasma. At least 13 missense mutations have been identified that are linked to type 2B vWD [47]. Most of them are clustered within a short sequence 540–578, a segment of the vWF A1 domain containing the putative inhibitor of vWF binding to GPIb [12]. Enhanced platelet agglutination at low ristocetin concentration provides a fairly specific screening assay to identify vWD type 2B [48]. Type 2M vWD refers to qualitative variants with decreased binding to platelets that is not caused by the absence of high-molecular-weight multimers. Type 2N vWD is due to failure of vWF to bind and protect procoagulant factor VIII; it is characterized by normal level and normal multimeric pattern of vWF but decreased level of factor VIII in the circulating blood. A revised classification of vWD that is based on differences in pa-
thophysiology has recently been recommended [49, 50].
Genotype and phenotype of vWF Type 1 vWD is often dominant, type 2 vWD may be dominant or recessive, and type 3 vWD is recessive. Quantitative deficiencies are caused by deletions, promoter mutations, nonsense mutations, and frame-shift mutations, rarely by missense mutations, whereas qualitative defects are usually associated with missense mutations and small in-frame deletions and insertions. Diagnosis of mild vWD frequently poses a problem, especially in type 1 vWD, a dominantly inherited bleeding disorder with apparently normal structure and function of vWF coded for by the normal allele. On the other hand, inheritance of a single null vWF allele is not associated with bleeding symptoms in most heterozygous persons. It is possible that the numerous polymorphisms established in the vWF gene influence the synthesis and function of vWF, thus affecting the phenotype of vWD. Furthermore, the genotype at other loci, e.g., AB0 blood type and Lewis locus, has been found to be associated with the level of plasma vWF [51, 52]. vWF is increased in various stressful situations such as physical exercise, inflammation, and pregnancy. Therefore, repeated testing of functional and antigenic levels of vWF in plasma may be required to establish the correct diagnosis of vWD. If one allele fails to produce a functional protein, it may be expected that vWF levels would be about 50% of normal. However, most families with type 2A vWD show a clear dominant pattern, frequently with functional vWF levels of less than 10% of normal. Since vWF is multimeric, mutant subunits that are highly susceptible to proteolytic degradation become incorporated into vWF multimers (Fig. 4). The heterozygous carriers of the protease-sensitive vWF are usually completely devoid of large multimers, and not only of the abnormal fraction, since the functionally relevant highmolecular-weight vWF is a protease-sensitive heteromultimer. Thus, mutation in one allele responsible for increased protease sensitivity of vWF dominantly affects the phenotype of vWD. It is conceivable that dysfunctional subunits coded by the abnormal allele produce heterodimers with normal subunits via disulfide linking of the carboxy terminal domains (Fig. 5). The resulting dimers are dysfunctional, in the sense that they cannot be assembled into multimers and secreted. This tentative model may explain the dominant inheritance of the type 2A vWD that is characterized by secretion from endothelial cells of abnormally small molecular forms of vWF. The presence of a defective “polymerization site” in only one half of vWF subunits is sufficient for complete deficiency of the very large, highly adhesive vWF multimers. Abnormal dimer polymerization may also be responsible for failure of 1-deamino-8-D-arginine vasopressin
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Fig. 4 Increased proteolytic degradation of vWF multimers in type 2A vWD. The vWF subunit consists of several structural domains (panel A). The platelet-binding site and one collagen-binding site are located in the A1 domain. Another collagen-binding site is situated in the A3 domain. Extremely slowly, the protease cleaves the peptide bond Tyr842–Met843 (white arrow) in the normal vWF subunit. In an abnormal subunit (panel B), the sensitivity to proteolytic cleavage is increased due to enhanced exposure of the cleavage site (black arrow). The proteolytic degradation of vWF multimers is accelerated (panel C), since they are composed of both resistant and sensitive subunits
domain. Mutations within the same codon resulting in Gly742 ] Arg and Gly742 ] Glu have even been shown to result in either decreased processing or increased proteolytic breakdown of highly polymeric vWF, respectively [45]. It is conceivable that various mutations in the A2 domain may affect the multimeric distribution by both impaired secretion of vWF multimers and enhanced sensitivity to the plasma protease. Similar considerations might be applied to the properties of the type 2B vWF variants with increased affinity for GPIb. In heterozygotes, large polymers containing both normal and abnormal subunits will exhibit an increased binding affinity for platelet receptors. The mode of inheritance of type 2B vWD will be dominant, due to spontaneous adsorption of heteropolymeric vWF to platelets. In contrast to types 2A and 2B vWD, the functional defect in type 2N vWD is not dependent on multimeric size: procoagulant factor VIII binds as a monovalent ligand to vWF. The abnormal binding of factor VIII is not affected by formation of heteropolymeric forms, and the trait is inherited in a recessive manner. DDAVP is relatively ineffective in patients with type 2N; its therapeutic effect may be of only a short duration. In spite of significant progress in our understanding of the synthesis, structure, and functions of vWF, the variable presentation of bleeding symptoms in patients with vWD remains poorly understood. Due to the high prevalence of vWD, there is a high probability of compound heterozygosity. Although the new classification helps in predicting the response to DDAVP, the therapeutic efficacy of DDAVP will often require appropriate testing of individual patients.
References
Fig. 5 Abnormal polymerization of dimers in type 2A vWD. Dimers are formed from normal subunits (white symbols) and abnormal subunits (black symbols), with a mutation preventing interdimeric linking of the amino terminal domains. In a heterozygous carrier of the defect, 25% each of normal and abnormal homodimer will be produced, together with 50% of mixed heterodimer. Abnormal homodimer cannot polymerize further. The linear growth of vWF multimers will be blocked following incorporation of a heterodimer on each end of the polymeric chain
(DDAVP) to correct the bleeding symptoms in type 2A vWD, since predominantly small molecular forms of vWF are released in response to DDAVP treatment. Most amino acid substitutions established in the protease-sensitive vWF variants and in abnormally polymerizing vWF variants are situated within the same stretch of the amino acid sequence denoted as the A2
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