Chapter 126: Von Willebrand Disease

In 1926, Eric von Willebrand described a bleeding disorder in 24 of 66 members of a family from the Åland Islands.¹ Both sexes were afflicted, and the bleeding time was prolonged despite normal platelet counts and normal clot retraction. von Willebrand distinguished this condition from the other hemostatic diseases known at the time and recognized its genetic basis, calling the disorder “hereditary pseudohemophilia,” but incorrectly characterizing the inheritance as X-linked dominant. von Willebrand’s confusion about the inheritance pattern was probably the result of, at least in part, the greater recognition of bleeding symptoms in women because of the hemostatic stresses of menstruation and parturition. The proband in the original family, Hjördis, was 5 years old at the time of von Willebrand’s initial evaluation and ultimately died at age 13 years during her fourth menstrual cycle. Four of Hjördis’ sisters died between the ages of 2 and 4 years, and deaths in the family were also noted during childbirth.

An apparently similar disorder was independently reported in the United States by Minot and others in 1928. The original family in the Åland Islands was reexamined by von Willebrand and Jürgens in 1933, leading to the conclusion that the defect in this disorder was caused by an impairment of platelet function. It was not until 1953 that Alexander and Goldstein demonstrated reduced levels of coagulation factor VIII (FVIII) in von Willebrand disease (VWD) patients, along with prolonged bleeding time. This observation was confirmed by others, including studies of the original von Willebrand pedigree by Nilsson and coworkers. In the late 1950s, Nilsson and coworkers demonstrated that a fraction of plasma referred to as “I-O” could correct the FVIII deficiency and normalize the bleeding time, indicating that the defect in VWD was a result of the deficiency of a plasma factor rather than an intrinsic platelet abnormality. Infusion of fraction I-O promptly increased the FVIII level in a hemophilic patient, while in VWD the FVIII level rose gradually, peaking at 5 to 8 hours. Fraction I-O prepared from a hemophilia A patient was also shown to correct the defect in VWD, demonstrating that these disorders were caused by deficiencies of distinct plasma factors (reviewed in Refs. 2 and 3).

It was not until 1971 that Zimmerman, Ratnoff, and Powell prepared the first antibodies against what was thought to be a highly purified form of FVIII.⁴ This FVIII-related antigen was found to be normal in hemophilia A patients but decreased in VWD. This puzzle was finally resolved with the demonstration that von Willebrand factor (VWF) and FVIII are closely associated, with more than 98 percent of the mass of the complex made up of VWF (see section The Function of von Willebrand Factor below). Thus, antibodies raised against this complex predominantly recognize VWF. The first direct assay of VWF function was based on the observation that the antibiotic ristocetin induced thrombocytopenia and the demonstration by Howard and Firkin⁵ that ristocetin-induced platelet aggregation (RIPA) was absent in some VWD patients. Weiss and coworkers⁶ used this observation to develop a quantitative assay for VWF function that remains a mainstay of laboratory evaluation for VWD to this day. In 1973, several groups succeeded in dissociating VWF from FVIII procoagulant activity.^7,8

Final proof that VWF and FVIII are independent proteins encoded by distinct genes came with the complementary DNA (cDNA) cloning of the two molecules in 1984 and 1985.^{9,10,11,12,13,14} These discoveries also marked the beginning of the molecular genetic era for the study of VWF and FVIII, leading to the identification of gene mutations in many patients with hemophilia and VWD, as well as considerable insight into the structure and function of these related proteins.

Table 126–1 summarizes the current nomenclature and terminology for FVIII and VWF. VWD is a heterogeneous disorder with more than 20 variants described. The previous complex and confusing classification has been consolidated and simplified into six distinct types,¹⁵ as summarized in Table 126–2. Type 3 VWD is associated with very low or undetectable levels of VWF and severe bleeding. Type 1 VWD is characterized by concordant reductions in FVIII activity, VWF antigen, and ristocetin cofactor activity, generally to the range of 20 to 30 percent of normal, but sometimes up to 50 percent of normal, in association with a normal VWF multimer distribution. Type 2 VWD is heterogeneous and further divided into four subtypes (2A, 2B, 2M, and 2N) by the nature of the VWF qualitative dysfunction. Type 2A VWD results from abnormal VWF secretion or proteolysis and is characterized by a disproportionately low level of ristocetin cofactor activity relative to VWF antigen and absence of large and intermediate-sized multimers. Type 2B VWD results from an abnormal VWF molecule with increased affinity for platelet glycoprotein Ib (GPIb), and can also be associated with reduced high-molecular-weight VWF multimers and thrombocytopenia. Functional abnormalities in VWF can also result in defective interactions with platelets, as in type 2M VWD, or decreased FVIII binding to VWF, designated type 2N VWD and characterized by mild to moderate FVIII deficiency. Many other subtypes have been reported, including platelet-type (pseudo-) VWD, which is actually an intrinsic platelet disorder caused by mutations in GPIb (Chap. 120). Finally, acquired forms of VWD also occur, such as in patients with antibodies to VWF or thrombocytosis secondary to myeloproliferative neoplasms, resulting in accelerated loss of circulating VWF.

VWF is synthesized exclusively in endothelial cells and megakaryocytes. The VWF monomer is assembled into higher-order multimers, a structure required for optimal adhesive function, and performs two major functions in hemostasis. First, VWF serves as the initial critical bridge between circulating platelets and the injured blood vessel wall, accounting for the apparent defect in platelet function and prolonged bleeding times historically observed in VWD patients. Second, VWF serves as the carrier in plasma for FVIII, ensuring its stability and localizing it to the initial platelet plug for participation in thrombin generation and fibrin clot formation (Chap. 113). This tight, noncovalent interaction between VWF and FVIII accounts for the copurification of these two molecules and the resulting initial confusion as to the origin of hemophilia and VWD. FVIII is encoded by the F8 gene on the X chromosome (Chaps. 113 and 123), while VWF is encoded by the VWF gene on human chromosome 12.

THE VON WILLEBRAND FACTOR GENE AND COMPLEMENTARY DNA

The VWF cDNA was initially cloned from endothelial cells,^11,12,13,14 and the corresponding gene mapped to the short arm of chromosome 12 (12p13.3).¹¹ The VWF mRNA is approximately 9 kb in length, encoding a primary translation product of 2813 amino acid residues with an estimated Mr of 310,000. Comparison of the primary peptide sequence obtained from plasma VWF¹⁶ with the VWF cDNA sequence established the prepropolypeptide nature of VWF.¹⁷ Prepropolypeptide VWF is composed of a 22-amino-acid signal peptide, a 741-amino-acid precursor polypeptide known as the VWF propeptide (VWFpp), and the mature subunit.^{11,17,18,19,20} Cleavage of the 741-amino-acid propeptide from the amino terminus produces the mature VWF monomer subunit of 2050 amino acids (Fig. 126-1).

Analysis of the VWF sequence identifies four distinct types of repeated domains: three A domains, three B domains, two C domains, and four D domains,^18,21 within which appear additional repeating motifs (schematic in Ref. 22). The first pair of D domains is tandemly arranged in the VWFpp, followed by a partial and full D domain at the N terminus of the mature subunit. The final complete D domain is separated by a segment of more than 600 amino acids containing the triplicated A domains. The repeated domain structure of VWF suggests that the gene may have evolved via a complex series of partial duplications, although exon structure is not highly conserved between homologous domains.

Comparison of the VWF amino acid sequence to other proteins identifies a superfamily of related proteins that share sequence similarity with the VWF A domains.²³ The common theme among these potentially evolutionarily related genes is a role in extracellular matrix or adhesive function. Consistent with this notion, VWF functional domains for binding to the platelet receptor GPIb and specific ligands within the extracellular matrix have been localized to the VWF A repeats. A potential relationship between the VWF C domains and portions of thrombospondin and procollagen has also been proposed.²⁴

The VWF gene spans 178 kb and is divided into 52 exons.²⁵ Exons range in size from 40 bases to 1.4 kb (exon 28). Exon 28 is unusually large, encoding the entire Al and A2 domains and containing most of the known type 2A and all of the type 2B VWD mutations. The concentration of these defects within one exon has facilitated the identification of human mutations responsible for these VWD variants (see “Molecular Genetics of von Willebrand Disease,” below). A partial, nonfunctional duplication of the VWF gene, termed a pseudogene, is located on human chromosome 22.²⁶ The pseudogene, known as VWFP1, duplicates the middle portion of the VWF structural gene, spanning exons 23 to 34 and the intervening noncoding sequences. VWFP1 is approximately 97 percent identical in sequence to the authentic VWF gene, indicating that it is of fairly recent evolutionary origin.²⁷ Gene conversion involving the VWFP1 pseudogene, possibly through recombination with the large homologous exon 28 sequence, has been proposed as a mechanism for introducing mutations into the VWF gene.^28,29,30,31

VWF is synthesized exclusively in megakaryocytes and endothelial cells and, as a result, has frequently been used as a specific histochemical marker to identify cells of endothelial cell origin. Although generally assumed to mark all endothelial cells, VWF is expressed at widely varying levels among endothelial cells, depending on the size and location of the associated blood vessel.^32,33 A careful survey in the mouse identified wide differences in the level of VWF mRNA, with 5 to 50 times higher concentrations in the lung and brain, particularly in small vessels, than in comparable vessels in the liver and kidney. In general, the higher levels of VWF mRNA and antigen were found in the endothelial cells of large vessels rather than in microvasculature, and in venous rather than arterial endothelial cells.³³

Specific DNA sequences within or near the proximal promoter of the VWF gene appear to be required for endothelial-specific gene expression,^{34,35,36,37,38,39} although it is likely that additional important regulatory elements exist outside of this region, some of which may lie at a great distance.⁴⁰ VWF is expressed in most, but not all, endothelial cells,⁴¹ and this vascular-bed specific gene expression program is likely a result of the concerted action of multiple regulatory elements. Endothelial VWF gene expression also appears to be upregulated by exposure to shear stress. The length of a polymorphic GT repeat in the proximal VWF promoter correlates with the magnitude of this response, and several other more distal DNA sequences are predicted to be involved in a shear stress response.⁴² However, this GT repeat does not appear to influence circulating VWF levels.⁴³

VON WILLEBRAND FACTOR BIOSYNTHESIS

The processing steps involved in the biosynthesis of VWF are similar in megakaryocytes and endothelial cells (reviewed in Refs. 46 and 47). VWF is first synthesized as a large precursor monomer polypeptide, depicted schematically in Fig. 126-1. VWF is unusually rich in cysteine, which accounts for 8.3 percent of its amino acid content. All cysteines in the mature VWF molecule are thought to be involved in disulfide bonds,⁴⁸ although these bonds may be exposed in circulating mature VWF by shear stress.⁴⁹ Pro-VWF monomers are assembled into dimers through disulfide bonds at both C termini, and only dimers are exported from the endoplasmic reticulum (ER).^48,50,51

Glycosylation begins in the ER, with 12 potential N-linked glycosylation sites present on the mature subunit and three on the propeptide. Extensive additional posttranslational modification of VWF occurs in the Golgi apparatus, including the addition of multiple O-linked carbohydrate structures, sulfation, and multimerization through the formation of disulfide bonds at the N termini of adjacent dimers. It is unusual for a protein to undergo extensive disulfide bond formation at this late stage, and this process appears to be catalyzed by disulfide isomerase activity present within the VWFpp.⁵² Mutations at either of two specific cysteines within the propeptide that are thought to be critical for disulfide isomerase activity, or a shift in the spacing between them, results in loss of multimer formation.⁵² An intermediate species with disulfide bonds between the propeptide and VWF D’D3 domain appears briefly in either the late ER or early Golgi,⁵³ which may position these domains for subsequent multimerization. The multimerization process appears to require the slightly acidic environment of the distal Golgi.⁵⁴ The VWFpp self-associates and may also serve to align VWF subunits for multimer assembly.⁵⁵ However, the propeptide facilitates multimer assembly even when coexpressed as a separate molecule from the mature VWF monomer.^56,57

Propeptide cleavage occurs late in VWF synthesis or just prior to secretion. Cleavage occurs adjacent to two basic amino acids, Lys-Arg at positions –2 and –1. An Arg at position –4 is also required for recognition by the intracellular protease responsible for propeptide cleavage.⁵⁸ Multimerization and propeptide cleavage are not linked to each other. The multimers secreted by cultured endothelial cells contain both pro-VWF and mature subunits,^59,60 and recombinant VWF with a point mutation inhibiting propeptide cleavage is still assembled into normal multimer structures.⁶¹ Although propeptide cleavage appears to occur primarily intracellularly, cleavage may also occur after secretion.

VWF is stored in tubular structures within the α-granules of platelets and within the Weibel-Palade bodies in endothelial cells^62,63 (reviewed in Ref. 64). These large VWF structures form by tubular packing of the VWF N-terminal domains within the secretory granules.⁶⁵ Weibel-Palade bodies are derived from the Golgi apparatus and are found in most endothelial cells, although the number varies considerably between endothelial cell beds. It has been shown that VWF and FVIII colocalize in storage granules. Although VWF is not required to traffic FVIII to platelets,⁶⁶ VWF appears to play a role in trafficking FVIII to Weibel-Palade bodies in endothelial cells.^67,68 Weibel-Palade bodies mature as they move to the periphery of the cell in an ordered process dependent on Rab proteins and Rab effector proteins, which act as chaperones and organizers of the various stages of Weibel-Palade body maturity and subsequent exocytosis (reviewed in Ref. 69). The transmembrane glycoprotein P-selectin is also found in the membranes of both the α-granule and the Weibel-Palade body.⁷⁰ The VWF D’D3 domain has been shown in vitro to associate with P-selectin and to be necessary for the recruitment of P-selectin to Weibel-Palade bodies.⁷¹ There appears to be heterogeneity within Weibel-Palade body populations both in relative content of VWF and P-selectin and in response to regulated secretion by different stimuli.⁷² In addition to VWF and P-selectin, the Weibel-Palade body also contains tissue-type plasminogen activator (t-PA), a thrombolytic secreted protein that also may be released distinctly from VWF,⁷³ and several other proteins that are known to participate in inflammation or angiogenesis (for a complete list of Weibel-Palade contents see Ref. 74).

VWF is secreted from endothelial cells continuously via constitutive and constitutive-like (or basal) pathways and upon stimulated release of storage granules via a classic regulated pathway.^46,75 Regulated secretion of VWF from its storage site in the Weibel-Palade body is triggered by a number of secretagogues, including thrombin,⁷⁶ fibrin,⁷⁷ histamine,⁷⁸ the C5b-9 complement complex,⁷⁹ and several inflammatory cytokines.⁸⁰ Recent in vitro data suggests that there may also be suppression of regulated VWF secretion by statins.^81,82 The secretagogue desmopressin acetate (DDAVP), a vasopressin analogue, is used clinically for its capacity to cause a marked release of VWF and FVIII in vivo by acting through type 2 vasopressin receptors to induce secretion from the Weibel-Palade bodies in endothelial cells.⁸³ Constitutive-like secretion of VWF occurs evenly at the luminal and abluminal surface, while regulated secretion from the Weibel-Palade body is highly polarized in the luminal direction (Fig. 126-2).^75,84 While constitutively secreted multimers are of relatively small size, the multimers stored within the Weibel-Palade body are the largest, most biologically potent form.^85,86 The VWF stored in platelet α-granules is also enriched for large multimers.⁸⁷ The N-terminal D domains appear to be required for VWF storage, with deletion of any of the individual domains resulting in constitutive secretion.^88,89 It also appears that cleavage of the VWFpp is required for efficient formation of storage granules.⁹⁰

The concentration of VWF in plasma is approximately 10 mcg/mL, with approximately 15 percent of circulating VWF localized to the platelet compartment.⁹¹ Marrow transplants between normal and VWD pigs demonstrate that platelet VWF is derived entirely from synthesis within marrow megakaryocytes and does not contribute to the normal plasma VWF pool.^92,93,94 These studies also demonstrate that both the plasma and the platelet VWF pools are required for full hemostasis, although the plasma pool appears to be more critical.

Plasma VWF is further processed in the circulation through cleavage by a specific protease, ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs–13), resulting in reduction in the size of the largest multimers (reviewed in Ref. 95). After regulated secretion in vitro, ultralarge VWF multimers may anchor to the endothelial cell surface via P-selectin^96,97 resulting in shear stress and VWF cleavage by ADAMTS13. The major proteolytic cleavage site maps to the peptide bond between Tyr1605 and Met1606 in the VWF A2 domain,⁹⁸ and recombinant VWF missing the A2 domain is resistant to proteolysis.⁹⁹ VWF carrying a subgroup of type 2A VWD mutations exhibits increased susceptibility to cleavage by this protease,¹⁰⁰ and this is the proposed mechanism for the selective loss of large VWF multimers in this group of patients (see “Molecular Genetics of von Willebrand Disease,” below). Increased VWF susceptibility to proteolysis by ADAMTS13 has also been described in a subset of type 1 VWD patients, but the clinical significance of this is unclear as increased proteolysis appears to only occur under certain conditions.¹⁰¹ Decreased ADAMTS13 activity, either because of congenital deficiency or acquired inhibitors, plays a central role in the pathophysiology of thrombotic thrombocytopenic purpura (Chap. 132).

THE FUNCTION OF VON WILLEBRAND FACTOR

VWF is a large multivalent adhesive protein that plays an important role in platelet attachment to subendothelial surfaces, platelet aggregation at sites of vessel injury, and stabilization of coagulation FVIII in the circulation. Not only is the interaction of VWF and FVIII important for the protection of FVIII from inactivation or degradation, FVIII bound to VWF may localize to cells and/or sites where it can more readily participate in the promotion of blood coagulation and/or thrombus formation.

VWF is required for the adhesion of platelets to the subendothelium, particularly at moderate to high shear force. VWF performs this bridging function by binding to two platelet receptors, GPIb and GPIIb/IIIa, as well as to specific ligands within the exposed subendothelium at sites of vascular injury (reviewed in Ref. 103). Binding of VWF to its platelet receptors generally does not occur in the circulation under normal conditions. However, the interaction of VWF with exposed ligands in the vessel wall, combined with high shear stress conditions, facilitates VWF binding to platelet GPIb and subsequent platelet adhesion and activation. Activation of platelets leads to the exposure of the GPIIb/IIIa complex, an integrin receptor that can bind to fibrinogen, VWF, and other ligands, to form the platelet–platelet bridges required for thrombus propagation. Platelet adhesion to VWF immobilized at a site of injury appears to be a two-step process, with the initial tethering of the rapidly moving platelet dependent on the VWF–GPIb interaction and subsequent firm adhesion occurring through GPIIb/IIIa after platelet activation.¹⁰⁴ VWF may also play a role in inflammation by directly interacting with leukocytes,¹⁰⁵ but the clinical significance of this observation is not clear.

Von Willebrand Factor Binding to the Vessel Wall

VWF binds to the vessel wall at sites of vascular endothelial injury (reviewed in Ref. 106). VWF binds to several different types of collagens, including types I through VI. Distinct binding domains for the fibrillar collagens, types I and III, have been localized to specific segments within the VWF A1 and A3 repeats (see Fig. 126-1),^107,108 and a potential third domain has been identified in the VWFpp.¹⁰⁹ Studies of recombinant VWF suggest that the A3 collagen-binding domain may be the most important.^110,111 VWF has also been shown to bind to the nonfibrillar collagen type VI, which is resistant to collagenase¹¹² and colocalizes with VWF in the subendothelium.¹¹³ Type VI collagen supports the binding of VWF under high shear through cooperative interactions between binding domains within the VWF A1 and A3 repeat.¹¹⁴ Although VWF binding has also been demonstrated to a number of other potential components of the subendothelium, including glycosaminoglycans,^115,116 sulfatides,¹¹⁷ and VWF itself,¹¹⁸ the biologic significance of these interactions remains to be demonstrated.

Von Willebrand Factor Binding to Platelets

VWF interacts with platelets both to mediate platelet aggregation and platelet localization to sites of vascular injury (reviewed in Ref. 106). Circulating VWF does not spontaneously interact with platelets, but once bound to an injured vessel wall VWF is subjected to higher shear stresses and a platelet-binding site in the VWF A1 domain is uncovered. VWF interacts with a receptor complex on the surface of platelets composed of the disulfide-linked GPIbα and GPIbβ chains noncovalently associated with GPIX and GPV. The binding site for VWF is within a 293-amino-acid segment at the N-terminus of GPIb and requires sulfation of several key tyrosine residues for optimal binding.¹¹⁹ The GPIb binding domain within VWF lies within the A1 segment, within the disulfide loop formed between the cysteine residues at 1272 (amino acid 509 in mature VWF) and 1458 (695) (see Fig. 126-1).^120,121 GPIb binding to the A1 domain enhances proteolysis of recombinant VWF fragments by ADAMTS13 and suggests a feedback mechanism for limiting thrombus propagation in vivo.¹²² Scanning mutagenesis studies of recombinant VWF characterized a number of amino acid residues within the VWF A1 domain that are critical for binding to GPIb and for interaction with botrocetin.¹²³ Several mutations were also identified that increase platelet binding, an effect similar to that of mutations associated with type 2B VWD (see “Molecular Genetics of von Willebrand Disease,” below). These natural and synthetic mutations cluster in a small area on the surface of the VWF A1 domain structure, as revealed by x-ray crystallographic studies.¹²⁴ The complexity of the VWF A1–GPIb interaction is evidenced by the ability of gain-of-function and loss-of-function VWF A1 mutants to counterbalance each other in mice.¹²⁵ The structure of the A1 domain closely resembles that of other previously studied A domains, including the VWF A3 domain.^126,127,128 The structure of GPIb in complex with the VWF A1 domain provides insight into the structural basis for the gain of function mutations associated with type 2B VWD.¹²⁹ The abundant plasma protein β ₂-glycoprotein I can bind the VWF A1 domain when VWF is structurally open to GPIbα binding. This may result in biologically relevant inhibition of the VWF-platelet interaction, as inhibitory anti–β ₂-glycoprotein I autoantibodies found in some patients with antiphospholipid antibody syndrome are associated with thrombosis.¹³⁰

Ristocetin binds to both VWF and platelets, but the mechanism by which it enhances the VWF/GPIb interaction is still poorly understood.^131,132 The snake venom botrocetin appears to induce GPIb binding through a different alteration in the VWF A1 domain and is also used to study this interaction.¹²⁸ Heparin binds the VWF A1 domain within the loop formed by the disulfide bond formed between the residues Cys1272 and Cys1458,¹³³ where it appears to competitively inhibit VWF binding to GPIb^134,135 and enhance VWF proteolysis by ADAMTS13 in vitro.¹³⁶ Although it has been suggested that this may account for hemorrhage not predicted by conventional heparin monitoring, the clinical significance of the VWF-heparin interaction is not clear.

The arg-gly-asp-ser (RGDS) sequence at amino acids 2507 to 2510 of the mature VWF subunit serves as the binding site within VWF for GPIIb/IIIa. The GPIIb/IIIa complex, also known as integrin α_IIbβ₃, is a member of the integrin family of cell surface receptors. GPIIb/IIIa undergoes a conformational change to a high-affinity ligand-binding state following platelet activation which, in addition to VWF, can bind a number of other adhesive proteins, including fibrinogen. Although VWF is present in blood at much lower concentrations than is fibrinogen, evidence suggests that VWF may be a critical ligand. VWF participates in platelet tethering and adhesion to fibrin under flow conditions,^104,137 where the C1C2 domains of VWF are required for fibrin binding.¹³⁸ An RGD sequence is also present in the VWFpp, although its functional significance is unknown.

The Interaction of von Willebrand Factor with Factor VIII

The noncovalent interaction between FVIII and VWF is required for the stability of FVIII in the circulation, as is evident from the FVIII levels of less than 10 percent that are observed in most severe VWD patients. Although each VWF subunit appears to carry a binding site for FVIII, the stoichiometry for the VWF–FVIII complex found in normal plasma is approximately one to two FVIII molecules per 100 VWF monomers.¹³⁹ FVIII bound to VWF is also protected from proteolytic degradation by activated protein C (reviewed in Ref. 140). FVIII also appears to increase the susceptibility of VWF to proteolysis by ADAMTS13 under shear conditions.¹⁴¹

The FVIII binding domain within VWF has been localized to the first 272 N-terminal amino acids of the mature subunit.¹⁴² In mice, expression of the VWF D’-D3 domains alone has been shown to be sufficient to stabilize FVIII levels.¹⁴³ Antibody studies suggest a particularly critical role for amino acids 841 to 859.^144,145 The mutations identified in patients with type 2N VWD, in which VWF binding to FVIII is specifically affected (see “Molecular Genetics of von Willebrand Disease,” below), are all clustered in this region, including the most common type 2N mutation Arg854Gln.¹⁴⁶ The corresponding binding site for VWF on FVIII includes an acidic region at the N-terminus of the light chain (residues 1669 to 1689)¹⁴⁷ and requires sulfation of Tyr1680 for optimal binding.¹⁴⁸ Overlay of VWD 2N mutations with the crystal structure of the VWF A1–FVIII domains shows the 2N mutations clustered in a dynamic VWF TIL’ domain (shown in Fig. 126-3), implicating a need for flexibility in this domain for normal FVIII binding.¹⁴⁹ Thrombin cleavage after Arg1689 in FVIII activates and releases FVIII from VWF. Thus, VWF may serve to efficiently deliver FVIII to the sites of clot formation, where it can complex with factor IXa on the platelet surface.

MOLECULAR GENETICS OF VON WILLEBRAND DISEASE

VWD is an extremely heterogeneous and complex disorder, with more than 20 distinct subtypes reported (referenced in Ref. 150). A large number of mutations within the VWF gene have been identified (see Fig. 126-3). However, because of both the genetic complexity of VWD and the practical considerations of VWF gene sequencing in most clinical settings, a VWF gene mutation is not required for the diagnosis of VWD.¹⁵¹ A list is maintained by a consortium of VWD investigators and can be accessed through the Internet at http://www.vwf.group.shef.ac.uk/. These findings form the basis for the simplified classification of VWD outlined in Table 126–2¹⁵¹ and used throughout this chapter. Types 1 and 3 VWD are defined as pure quantitative deficiencies of VWF that are either partial (type 1) or complete (type 3). Type 2 VWD is characterized by qualitative abnormalities of VWF structure and/or function. The quantity of VWF found in type 2 VWD may be normal, but it is usually mildly to moderately decreased (see Table 126–2).

The diagnosis of VWD, particularly type 1 VWD, can be confounded by the incomplete penetrance of the disease and the wide range of VWF levels in normal populations (see “Laboratory Features” below). Nonpathogenic variation can impact laboratory assays in vitro, as is the case with Asp1472His, which alters VWF-ristocetin interactions but has no impact on hemostasis in vivo.¹⁵² Ethnicity should also be considered. European ancestries were overrepresented in the studies which informed laboratory cutoffs, while African Americans exhibit generally higher VWF and FVIII levels. Additionally, there are numerous VWF gene “mutations” previously thought to be causative of VWD which are now known to be common in African Americans^153,154 who have normal VWF and FVIII levels,¹⁵³ including the Asp1472His variant.¹⁵⁵

Type 1 von Willebrand Disease

Type 1 VWD is the most common form, accounting for approximately 70 percent of patients with the disease. Type 1 VWD is generally autosomal dominant in inheritance and is associated with coordinate reductions in FVIII, ristocetin cofactor activity, and VWF antigen with maintenance of the full complement of multimers (Fig. 126-4). Subgroups within type 1 VWD have been proposed based on the relative levels of VWF present in the plasma and platelet pools,^{156,157,158,159} but with the exception in some circumstances for an accelerated VWF clearance phenotype (unofficially termed VWF type 1C¹⁶⁰), subtype distinctions in type 1 VWD are not generally used in clinical practice.

Type 1 VWF was previously assumed to simply represent the heterozygous form of type 3 VWD. However, in a large Canadian study, 48 percent of heterozygous carriers of type 3 VWD gene mutations carried a diagnosis of type 1 VWD, while the remainder were asymptomatic.¹⁶¹ Furthermore, in two large studies of type 1 VWD families, numerous putative VWF mutations were identified, but very few were predictive of VWF null alleles.^31,162 Thus, some, but probably not all (see section Clinical Features below), type 1 VWD is the result of defects within the VWF gene. Studies of type 1 VWD mutations in patients, in vitro, and in animal models have characterized diverse mechanisms underlying type 1 VWD, including decreased VWF production,¹⁶³ retention of VWF in the ER,^164,165 impaired VWF secretion,^165,166 and decreased VWF survival.^163,166,167 Mutations that give rise to defective VWF subunits that interfere in a dominant negative way with the normal allele may be particularly likely to cause symptomatic VWD in the heterozygote.¹⁶⁸ For example, mutations at several cysteine residues in the VWF D3 domain and in the VWFpp of patients with moderately severe type 1 VWD. VWF carrying one of these mutations is retained in the ER, where it is proposed to exert a dominant negative effect on VWF derived from the normal allele via heterodimerization and degradation.^169,170

To date, most mutation studies and genetic linkage analysis of type 1 VWD have been consistent with defects within the VWF gene. Although no single mutation can explain the majority of type 1 VWD, common VWF founder mutations can occur within populations, such as Tyr1584Cys identified in 14 percent of Canadian type 1 VWD patients, and possibly a similar proportion of patients in Europe.^162,171 The Tyr1584Cys mutation is associated with decreased VWF survival, likely a result of increased susceptibility to proteolysis by ADAMTS13.^{172,173,174,175} These large multicenter studies of type 1 VWD families found candidate VWF mutations in 63 to 70 percent of families with type 1 VWD, leaving 37 to 40 percent of type 1 VWD index cases without a putative mutation in VWF.^162,171 Cases with VWF gene mutations tend to be more severe and highly heritable, while cases without an identifiable VWF mutation generally have higher VWF antigen (VWF:Ag) levels (>30 IU/dL).³¹ In a large, multicenter European study of 150 type 1 VWD families, about one-third of cases historically diagnosed to have type 1 VWD were found to have abnormal multimers, and of these nearly all (95 percent) had a putative VWF gene mutation and significantly lower VWF:Ag, VWF ristocetin cofactor activity (VWF:RCo), assay of FVIII activity (FVIII:C), and VWF collagen-binding assay (VWF:CB) levels. Conversely, index cases with normal multimers had higher laboratory VWF values and fewer identifiable VWF mutations (55 percent), suggesting that the pathogenic mechanism(s) underlying this cohort of “true” type 1 VWD patients is more genetically complex.¹⁶² Given the complex biosynthesis and processing of VWF, defects at a number of other loci could also be expected to result in quantitative VWF abnormalities (reviewed in Ref. 176). This concept is supported by families with type 1 VWD in which bleeding histories and low ristocetin cofactor activities do not always cosegregate with genetic markers at the VWF locus,^31,177 while one or more genetic factors outside the VWF locus may be associated with the variation in bleeding severity observed within VWD pedigrees.^178,179 It is interesting to note that a spontaneous mouse model of type 1 VWD exhibits up to 20-fold reductions in plasma VWF as a result of an unusual mutation in a glycosyltransferase gene, leading to aberrant posttranslational processing of VWF and accelerated clearance from plasma.¹⁸⁰ Similar mechanisms affecting VWF survival, perhaps combined with altered proteolysis,^181,182,183 may explain the observed modifying effect of the ABO blood group glycosyltransferases on plasma VWF survival.¹⁸⁴ Additional genetic factors have been implicated to influence VWF via altered survival, including the clearance receptors CLEC4M and LRP1 (CD91) (reviewed in Ref. 185). The biologic consequences of VWF modifiers identified in normal populations are unclear, and studies are needed to determine their significance in VWD.

Type 3 von Willebrand Disease

Patients with type 3 VWD account for 1 to 5 percent of clinically significant VWD, have very low or undetectable levels of plasma and platelet VWF:Ag and VWF:RCo, and generally present early in life with severe bleeding.¹⁸⁶ FVIII coagulant activity is markedly reduced but usually detectable at levels of 3 to 10 percent of normal. Type 3 VWD has generally been considered an autosomal recessive disorder, but in a recent Canadian study of 100 individuals in 34 families, 48 percent of “carriers” had a diagnosis of type 1 VWD,¹⁶¹ suggesting the dominant type 1 VWD pattern of inheritance is common in type 3 VWD families.

Mutations associated with type 3 VWD have been reported throughout the VWF gene (http://www.vwf.group.shef.ac.uk/). Gross VWF gene deletion detectable by Southern blot^{26,187,188,189,190} or multiple ligation-probe amplification^161,191 is the molecular mechanism for type 3 VWD in only a small subset of families. However, large deletions may confer an increased risk for the development of alloantibodies against VWF.^26,189 A similar correlation between gene deletion and risk for alloantibody formation has been observed in hemophilia (Chap. 123). Comparative analysis of VWF genomic DNA and platelet VWF mRNA has identified nondeletion defects resulting in complete loss of VWF mRNA expression as a molecular mechanism in some patients with type 3 VWD.^192,193 A number of nonsense and frameshift mutations that would be predicted to result in loss of VWF protein expression or in expression of a markedly truncated or disrupted protein have been identified in some type 3 VWD families.^{168,194,195,196} A frameshift mutation in exon 18 appears to be a particularly common cause of type 3 VWD in the Swedish population and has been shown to be the defect responsible for VWD in the original Åland Island pedigree.^197,198 This mutation results in a stable mRNA encoding a truncated protein that is rapidly degraded in the cell.¹⁹⁹ This mutation also appears to be common among type 3 VWD patients in Germany,²⁰⁰ but not in the United States.²⁰¹

Type 2A von Willebrand Disease

Type 2A is the most common qualitative variant of VWD and is generally associated with autosomal dominant inheritance and selective loss of the large and intermediate VWF multimers from plasma (see Fig. 126-4). A 176-kDa proteolytic fragment present in normal individuals is markedly increased in quantity in many type 2A VWD patients. This fragment is consistent with proteolytic cleavage of the peptide bond between Tyr1605 and Met1606.^98,202 Based on this observation, initial DNA sequence analysis in patients centered on VWF exon 28, in the region encoding this segment of the VWF protein, leading to the identification of the first point mutations responsible for VWD.²⁰³ Since that time, a large number of mutations have been identified, accounting for the majority of type 2A VWD patients.¹⁹⁴ Many of these mutations are clustered within a 134-amino-acid segment of the VWF A2 domain (between Gly1505 and Glu1638; see Fig. 126-3), and the most common, Arg1597Trp, appears to account for about one-third of type 2A VWD patients.^194,195,204

Type 2A VWD mutations have been grouped by two distinct molecular mechanisms. In the first subset, classified as group 1, the type 2A VWD mutation has been commonly considered a defect in intracellular transport, with retention of mutant VWF in the ER. In addition to retention or degradation of mutant VWF in the ER, type 2A mutations can also disrupt intracellular processing and secretion via defective multimerization and/or loss of regulated storage.²⁰⁵ In the second subset, or group 2, mutant VWF is normally processed and secreted in vitro, and thus loss of multimers in vivo is presumed to occur based on increased susceptibility to proteolysis in plasma^{98,206,207,208,209} at the Tyr1605-Met1606 site cleaved by ADAMTS13.^101,210 The susceptibility of type 2A VWD mutations to proteolysis by ADAMTS13 in vitro supports accelerated proteolysis as a mechanism for the loss of high-molecular-weight VWF multimers in these patients.²⁰⁴

The multimer structure of platelet VWF correlates well with the underlying type 2A mechanisms. Group 1 patients show loss of large VWF multimers within platelets as a result of defective synthesis, while group 2 patients have normal VWF multimers within the protected environment of the α granule.²⁰⁶ These observations confirm the earlier subclassification of type 2A VWD based on platelet multimers.¹⁵⁶ Subclassification into group 1 or 2 might be expected to predict response to DDAVP therapy, although this remains to be demonstrated.

In addition to the major classes of type 2A VWD described above, a number of rare variants historically classified as types IIC to IIH, type IB, and “platelet discordant” are included in the more general type 2A category. Most of these rare variants were distinguished on the basis of subtle differences in the multimer pattern (see Fig. 126-4; multimer changes relative to the location of type 2 mutations is reviewed in Ref. 211). The IIC variant is usually inherited as an autosomal recessive trait and is associated with loss of large multimers and a prominent dimer band. Several mutations have been identified in the VWFpp of these patients,^212,213,214 presumably interfering with multimer assembly and/or trafficking to storage granules. A mutation at the C terminus of VWF, interfering with dimer formation, was described in a patient with the IID variant.²¹⁵ Most of the other reported variants of type 2A VWD are quite rare, often limited to single case reports.

Type 2B von Willebrand Disease

Type 2B VWD is usually inherited as an autosomal dominant disorder and is characterized by thrombocytopenia and loss of large VWF multimers. The plasma VWF in type 2B VWD binds to normal platelets in the presence of lower concentrations of ristocetin than does normal VWF and can aggregate platelets spontaneously. Accelerated clearance of the resulting complexes between platelets and the large, most adhesive forms of VWF accounts for the thrombocytopenia and the characteristic multimer pattern (see Fig. 126-4).

The peculiar functional abnormality characteristic of type 2B VWD suggested a molecular defect within the GPIb binding domain of VWF. For this reason, initial DNA sequence analysis focused on the corresponding portion of VWF exon 28.^216,217 Type 2B mutations are located within the VWF A1 domain at one surface of the described crystallographic structure.^124,129 The four most common mutations are clustered within a 36-amino-acid stretch between Arg1306 and Arg1341 (see Fig. 126-3); together, these account for more than 80 percent of type 2B VWD patients.¹⁹⁵ Functional analysis of mutant recombinant VWF^{218,219,220,221,222} confirms that these single-amino-acid substitutions are sufficient to account for increased GPIb binding and the resulting characteristic type 2B VWD phenotype. Structural studies of type 2B VWD mutations show that these residues interact with the leucine rich repeats of GPIb thought to be critical to the VWF A1–GPIb interactions under shear.²²³ Type 2B mutations have now been modeled extensively in mice, all of which exhibited accelerated VWF clearance, as expected.²²⁴ Type 2B VWD mice also had short-lived platelets, with evidence of macrophage-mediated platelet clearance.²²⁵ In these models, platelets were observed to be coated by type 2B VWF,²²⁵ a phenomenon that may contribute to a previously unsuspected acquired platelet function defect.²²⁶ Interestingly, mice with the same type 2B mutations exhibit variable loss of large multimers²²⁴ and varying degrees of thrombocytopenia,²²⁷ similar to the variation observed in human pedigrees. Individual type 2B patients can also exhibit varying multimer structure and platelet counts over time. For example, two siblings with the Arg1306Trp mutation and abnormal multimers intermittently regained normal VWF multimer distribution during periods of thrombocytopenia.²²⁸

Families have been described that exhibit enhanced VWF binding to GPIb but a normal distribution of VWF multimers. These variants, previously referred to as type I New York, type I Malmö, and type I Sydney, are now all designated as type 2B VWD. Type I New York and type I Malmö are caused by the same VWF mutation, Pro1266Leu. This mutation is located within the cluster of type 2B mutations in the VWF A1 domain and results in a similar increase in platelet GPIb binding.²²⁹

Type 2N von Willebrand Disease

As described in Chap. 123, hemophilia A results from defects in the FVIII gene and is inherited in an X-linked recessive manner. Distinct from hemophilia A, families have been reported in which the inheritance of low FVIII appeared to be autosomal, based on the occurrence of affected females or direct transmission from an affected father.^230,231 Several cases of an apparent autosomal recessive decrease in FVIII were shown to be caused by decreased VWF binding of FVIII,^232,233,234 now referred to as VWD type 2N, after the Normandy province of origin of the first patient. DNA sequence analysis has identified more than 37 distinct mutations²³⁵ associated with this disorder, most located at the VWF N terminus (see Fig. 126-3) (curated in the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis VWF Database, http://www.vwf.group.shef.ac.uk/). One of these mutations, Arg854Gln, appears to be particularly common, may contribute to variability in the severity of type 1 VWD in some cases,²³⁶ and may also cause a VWF secretion defect.²³⁷ Rare cases of misdiagnosis of type 2N have led to treatment with recombinant FVIII for presumed hemophilia A, with poor responses and adverse clinical outcomes.²³⁸

Type 2M von Willebrand Disease

This category was classically reserved for rare VWD variants in which a defect in VWF platelet-dependent function leads to significant bleeding but VWF multimer structure is not affected (although some have subtle multimer abnormalities). Most contemporary type 2M variants are indeed associated with absent ristocetin cofactor activity but normal platelet binding with other agonists. A total of 28 type 2M VWD mutations have been described,²³⁵ including a number of other families with normal VWF multimers and disproportionately decreased ristocetin cofactor activity,^239,240 families with a combination of defects in VWF:CB and VWF–GPIb interactions of varying severities,^241,242 and mutations with isolated defects in VWF:CB with normal VWF:RCo activity.²³⁵ Several families have also been described with a VWD variant (VWD Vicenza) characterized by larger-than-normal VWF multimers and classified as either type 1 or type 2M VWD.²⁴³ Genetic linkage analysis indicates that the Vicenza defect lies within the VWF gene,²⁴⁴ and mutations within the VWF gene have been reported to be associated with VWD Vicenza.²⁴⁵ The underlying molecular mechanism responsible for the VWD Vicenza phenotype remains controversial,²⁴⁶ although recent kinetic modeling suggests that altered VWF survival alone could account for the VWF perturbations observed in this disorder.²⁴⁷

INHERITANCE

Type 1 VWD is generally transmitted as an autosomal dominant disorder, and accounts for approximately 70 percent of clinically significant VWD. However, disease expressivity is variable, and penetrance is incomplete.¹⁶⁸ Laboratory values and clinical symptoms can vary considerably, even within the same individual, and establishing a definite diagnosis of VWD is often difficult. In two large families with type 1 VWD, only 65 percent of individuals with both an affected parent and an affected descendent had significant clinical symptoms.²⁴⁹ For comparison, 23 percent of the unrelated spouses of the patients, who presumably did not have a bleeding disorder, were judged to have a positive bleeding history.

A number of factors have long been known to modify VWF levels, including ABO blood group, secretor blood group, estrogens, thyroid hormones, age, and stress.^250,251,252 ABO blood group is the best characterized of these factors. Genome-wide linkage has repeatedly confirmed strong linkage between the ABO locus and VWF levels (reviewed in Ref. 253). Mean VWF:Ag levels are approximately 75 percent for type O individuals and 123 percent for type AB individuals when compared to a pool of normal donor plasmas. Thus, it may be difficult to differentiate between a low-normal VWF value and mild type 1 VWD in blood group O individuals. In recent years, additional modifiers of VWF have been identified in large genetic association studies,^254,255 including genes associated with VWF intracellular trafficking (STXBP5^256,257) and VWF clearance (CLEC4M²⁵⁸). Additionally, a genome-wide association study identified a novel genetic locus on chromosome 2 contributing to variation in plasma VWF.²⁵⁹ The variable expressivity and incomplete penetrance of type 1 VWD and overlap in VWF levels between mild type 1 VWD and normal populations has complicated the determination of accurate incidence figures for VWD, with estimates ranging from as high as 1 percent^260,261 to as low as 2 to 10 per 100,000 population.²⁶²

In general, the type 2 VWD variants, which comprise 20 to 30 percent of all VWD diagnoses,²⁶³ are more uniformly penetrant. Type 2A and type 2B VWD account for the vast majority of patients with qualitative VWF abnormalities. Types 2A, 2B, and 2M are generally autosomal dominant in inheritance, although Type 2N and other rare cases of apparent recessive inheritance have been reported.

Estimates of prevalence for severe (type 3) VWD range from 0.5 to 5.3 per 1,000,000 population.^264,265,266 Although this variant is frequently defined as autosomal recessive in inheritance, this is not a consistent finding. As described above, one or both parents of a severe VWD patient can be clinically asymptomatic and have entirely normal laboratory test results, although in many families one or both parents appear to be affected with classic type 1 VWD. Thus, in some families, severe VWD may represent the homozygous form of type 1 VWD. In this model, the apparent recessive inheritance in a subset of families could simply be the result of the incomplete penetrance of type 1 VWD. Alternatively, there may be a fundamental difference in the molecular mechanisms responsible for type 1 and type 3 VWD.¹⁶⁸

Compound heterozygosity (the presence of more than one VWF gene mutation) can occur, and the clinical presentation in such cases can depend on the interaction between the different mutant VWF proteins. Compound heterozygosity can impact response to therapy because of a complex VWD phenotype and has implications for genetic counseling. If compound heterozygosity is deduced from the family history and/or laboratory studies or discovered during genetic testing, the most recent update to the VWD nomenclature represents both types separated by a slash (/), such as VWD type 2B/2N.¹⁵¹

CLINICAL SYMPTOMS

Mucocutaneous bleeding is the most common symptom in patients with type 1 VWD.²⁴⁹ It is important to note that more than 20 percent of normal individuals may give a positive bleeding history.²⁶⁷ Bleeding assessment scores have evolved over many years,²⁶⁸ leading the International Society on Thrombosis and Haemostasis to propose a unified Bleeding Assessment Test²⁶⁹ for research purposes. Although high bleeding scores are suggestive of a bleeding diathesis and can predict future bleeding,²⁷⁰ no bleeding questionnaire as yet is clearly diagnostic for VWD. These observations, together with the limited sensitivity and specificity of the currently available laboratory tests (see below), makes the diagnosis of mild VWD quite difficult and probably contributes to the wide range of prevalence figures for type 1 VWD currently in the literature. A National Heart Lung and Blood Institute Expert Panel has proposed clinical guidelines for evaluating patients to determine whether laboratory testing for VWD or other bleeding disorders is warranted.¹⁵⁰

Epistaxis occurs in approximately 60 percent of type 1 VWD patients, 40 percent have easy bruising and hematomas, 35 percent have menorrhagia, and 35 percent have gingival bleeding. Gastrointestinal bleeding occurs in approximately 10 percent of patients.¹⁵⁰ An apparent association between hereditary hemorrhagic telangiectasia (HHT) and VWD has been reported in several families. The causative genes in HHT were identified and are located on chromosomes 9q33–34, and 12q13²⁷¹ (Chap. 122), distinct from the VWF gene on chromosome 12p13. However, because inheriting VWD is likely to increase the severity of bleeding from HHT, the diagnosis is more likely to be made in patients inheriting both defects.²⁷² Mucocutaneous bleeding is common after trauma, with approximately 50 percent of patients reporting bleeding after dental extraction, approximately 35 percent after trauma or wounds, 25 percent postpartum, and 20 percent postoperatively. Hemarthroses in patients with moderate disease are extremely rare and are generally only encountered after major trauma. The bleeding symptoms can be quite variable among patients within the same family and even in the same patient over time. An individual may experience postpartum bleeding with one pregnancy but not with others, and clinical symptoms in mildly to moderately affected type 1 individuals often ameliorate by the second or third decade of life. Aside from an infrequent type 3 patient, death from bleeding rarely occurs in VWD.

Thrombocytopenia is a common feature of type 2B VWD and is not seen in any other form of VWD. Most patients only experience thrombocytopenia at times of increased VWF production or secretion, such as during physical effort, in pregnancy, in newborn infants, postoperatively, or if an infection develops. The platelet count rarely drops to levels thought to contribute to clinical bleeding.^273,274 Infants with type 2B VWD may present with neonatal thrombocytopenia, which could be confused with neonatal alloimmune thrombocytopenia, neonatal sepsis, or congenital thrombocytopenia.

Patients who are homozygous or compound heterozygous for type 2N VWD generally have normal levels of VWF:Ag and VWF:RCo and normal VWF platelet adhesive function. However, FVIII levels are moderately decreased, resulting in a mild to moderate hemophilia-like phenotype.¹⁴⁶ In contrast to patients with classic hemophilia A (FVIII deficiency), these patients do not respond to infusion of purified FVIII and should be treated with VWF-containing concentrates.²⁷⁵ Heterozygotes for this disorder may have mildly decreased FVIII levels but are generally asymptomatic. Although type 2N VWD appears to be considerably less common than classic hemophilia A, it should be considered in the differential diagnosis of FVIII deficiency, particularly if any features suggest an autosomal pattern of inheritance. Although the FVIII level rarely drops below 5 percent, type 2N VWD mutation can be associated with FVIII levels as low as 1 percent, when co-inherited with a type 3 VWD allele.²⁷⁶ The latter observation further suggests that a diagnosis of type 2N VWD should also be considered in patients with marked reductions of FVIII.

Patients with type 3 VWD can suffer from severe clinical bleeding and experience hemarthroses and muscle hematomas, as in severe hemophilia A (Chap. 123). After infusion of VWF-containing plasma fractions, some of these patients develop anti-VWF antibodies that neutralize VWF (reviewed in Ref. 277).

Other heritable coagulopathies can coexist with VWF deficiency. An evaluation for other factor deficiencies or platelet disorders should be considered in patients that have a suggestive family history, a bleeding phenotype out or proportion or inconsistent with an expected VWD pattern, or a poor response to therapy. In VWD patients with combination coagulopathies, treatment of both disorders may be necessary to achieve a good clinical result.²⁷⁸

In the initial laboratory evaluation of patients suspected by history of having VWD, the following tests are routinely performed: assay of FVIII:C, VWF:Ag, and VWF:RCo. Other tests that are commonly used include RIPA, VWF:CB, and VWF multimer analysis. Routine coagulation studies, such as prothrombin time (PT) or activated partial-thromboplastin time (aPTT), are generally not useful in the evaluation of VWD. However, the aPTT can be prolonged in subjects with VWF deficiency,²⁷⁹ or in patients with homozygous type 2N VWD, because of the reduction in FVIII level. The wide range of normal and the considerable overlap with the levels observed in type 1 VWD make borderline levels difficult to interpret. A variety of concurrent diseases and drugs may modify the results of individual tests. Many conditions, such as recent exercise, age, pregnancy, time of the menstrual cycle, estrogen therapy, hypo- or hyperthyroidism, diabetes, uremia, liver disease, infection, myeloproliferative neoplasms, or malignancy can affect the FVIII activity, VWF:Ag, and ristocetin cofactor activity levels. These values can be regarded as acute-phase reactants, and even minor illnesses can increase the levels in a VWD patient to normal. Appropriate processing of laboratory specimens is also critical as VWF parameters can be artifactually skewed (either high or low) by phlebotomy conditions or specimen handling (reviewed in Ref. 150). Even controlling for many of these factors, the coefficients of variation of repeated VWF:Ag and ristocetin cofactor assays in a single person are quite large,²⁸⁰ and can be influenced by numerous factors including diurnal variation.²⁸¹ For this reason, repeated measurements are usually necessary, and the diagnosis of VWD or its exclusion should generally not be based on a single set of laboratory values.

The laboratory diagnosis of type 1 VWD can be confounded by the wide range of VWF levels in “normals” and borderline laboratory results. An alternative strategy is to classify some patients for whom the diagnosis of VWD is ambiguous as “low VWF,” recognizing that these patients may have an increased risk of bleeding without labeling them as type 1 VWD.^282,283 In response to this need to distinguish those patients with VWD from nonbleeding individuals with moderately low levels of VWF (30 to 50 IU/dL), a threshold of less than 30 IU/dL has been recommended.¹⁵⁰ In clinical practice there remains wide variation in the assignment of normal VWF ranges and in the interpretation of laboratory results to make a VWD diagnoses.^284,285,286

FACTOR VIII

FVIII levels in VWD patients are generally coordinately decreased along with plasma VWF, although skewing of FVIII-to-VWF ratios can be observed.²⁸⁷ Levels in type 3 VWD generally range from 3 to 10 percent. In contrast, the levels in type 1 and the type 2 VWD variants (other than 2N) are variable and usually only mildly or moderately decreased. The FVIII level in type 2N VWD is more severely decreased, but rarely to less than 5 percent.

VON WILLEBRAND FACTOR ANTIGEN

Plasma VWF:Ag is usually quantitated by electroimmunoassay or an enzyme-linked immunosorbent assay (ELISA) technique. In type 1 VWD, the VWF:Ag assay usually parallels VWF:RCo, but it has lower specificity and sensitivity than the VWF:RCo assay. In patients with type 2 VWD, the VWF:Ag is variably decreased but can be normal (see Table 126–2).

RISTOCETIN COFACTOR ACTIVITY

The standard measure of VWF activity, the VWF:RCo quantitates the ability of plasma VWF to agglutinate platelets via platelet membrane GPIbα in the presence of ristocetin.²⁸⁸ In the most common method, normal platelets washed free of plasma VWF are used either as fresh platelets or after formaldehyde fixation. This assay has long been reported to be the most sensitive and specific single test for the detection of VWD.²⁸⁹ Numerous alternative methods have been proposed as adjuncts or replacements of the standard platelet-based ristocetin cofactor activity assay. However, none as yet can serve as a surrogate for VWF:RCo (reviewed in Ref. 290).

Ristocetin cofactor activity is generally decreased coordinately with VWF:Ag and FVIII in type 1 VWD patients. In type 2 VWD variants, ristocetin cofactor activity can be disproportionately decreased, as is usually the case in type 2A variants (and sometimes type 2B), because of the greater dependence of the ristocetin-mediated platelet–VWF interaction on the presence of larger VWF multimers, and in type 2M, because of decreased VWF-platelet interactions (see Table 126–2). Thus, the VWF:RCo-to-VWF:Ag ratio has been proposed as a means to distinguish between type 1 and type 2 VWD, with a ratio of VWF:RCo-to-VWF:Ag of less than 0.7 being indicative of a qualitative (type 2) VWF defect.¹⁵¹ However, in patients with very low VWF:Ag levels this ratio may not be reliable because of the limits of sensitivity of most VWF:RCo assays. The VWF:RCo assay is uninformative in the presence of the VWF Asp1472His variant, which interferes with the VWF–ristocetin interaction in vitro.¹⁵⁵

RISTOCETIN-INDUCED PLATELET AGGLUTINATION

Similar to the ristocetin cofactor assay above, the RIPA assay also measures platelet agglutination caused by ristocetin-mediated VWF binding to platelet membrane GPIbα. In the case of RIPA, ristocetin is added directly to patient platelet-rich plasma and platelet aggregation is measured. Hyperresponsiveness to RIPA results either from a type 2B VWD mutation or an intrinsic defect in the platelet (platelet-type or pseudo-VWD). In these disorders, patient platelet-rich plasma agglutinates spontaneously or at low ristocetin concentrations of only 0.2 to 0.7 mg/mL. At these concentrations, normal platelet-rich plasma does not agglutinate. Type 2B and platelet-type VWD can be distinguished by RIPA experiments performed with separated patient platelets or plasma mixed with the corresponding component from a normal individual or paraformaldehyde-fixed platelets. The RIPA is generally reduced in most other subtypes of VWD (see Table 126–2).

MULTIMER ANALYSIS

Analysis of plasma VWF multimers is critical for the proper diagnosis and subclassification of VWD (see Fig. 126-4). This is generally accomplished by agarose gel electrophoresis of plasma VWF to separate VWF multimers on the basis of molecular size, with the largest multimers migrating more slowly than the intermediate or smaller multimers. The multimers may be visualized by autoradiography after incubation with ¹²⁵I-monospecific antihuman VWF antibody or, more commonly, by nonradioactive immunologic techniques. The normal multimeric distribution is an orderly ladder of major protein bands of increasing molecular weight, going from the smallest to the largest VWF multimers (see Fig. 126-4). Each normal multimer has a fine structure consisting of one major component and two to four satellite bands.²⁹¹ Type 2B and most of the type 2A variants were initially distinguished from each other on the basis of subtle variations in the satellite band pattern. In a large European multicenter type 1 VWD study, careful analysis of VWF multimers in subjects historically diagnosed as type 1 VWD, including patients diagnosed at experienced centers, found one-third of “type 1” VWD patients had subtly abnormal multimers.²⁹² Although this previously would have required reclassification of these patients as type 2 VWD, the most recent update on the classification of VWD by the International Society on Thrombosis and Haemostasis Subcommittee on von Willebrand Factor expanded the category of type 1 VWD to permit subtle VWF multimer abnormalities.¹⁵¹ The authors of the European type 1 VWD study note that having samples from the index case, affected family members, and unaffected family members on one gel made qualitative defects more readily detectable, and that intermediate-resolution multimer gels were superior to low-resolution multimer gels in detecting abnormalities in this population. Use of VWF tests sensitive to VWF multimer structure have been proposed by some experts as proxies for VWF multimer testing, such the VWF:RCo-to-VWF:Ag ratio (VWF RCo:Ag ratio) or VWF:CB assay.²⁹³ In studies comparing these approaches as surrogates for VWF multimer assays, the (VWF RCo:Ag ratio) ratio was found to be less sensitive than multimer gel techniques in identifying qualitative VWF defects,²⁹² while VWF:CB an detect some type 2M VWD patients who have normal VWF multimers.^294,295 These observations support a continued important role for VWF multimer analysis in the laboratory evaluation of VWD.

ADDITIONAL LABORATORY TESTS

As a result of the variable sensitivity and specificity of laboratory testing for VWD, additional diagnostic studies may be useful in the classification of VWD patients. The VWF:CB measures VWF binding to collagen (type I, type III, type VI, or mixed) by ELISA. As above, assays based upon VWF:CB can complement the VWF:RCo in detecting type 2 VWD variants,^{296,297,298,299} and an abnormal VWF:CB-to-VWF:Ag ratio (VWF CB:Ag ratio) is suggestive of a qualitative VWF defect.¹⁵¹ Abnormalities in VWF:CB can reflect loss of high-molecular-weight multimers and/or the discrete loss of collagen binding caused by a type 2M mutation. Use of VWF:CB assays is expanding in clinical practice, with select sites including this test routinely in initial VWD diagnostic laboratory testing.

Another group of tests are included in the term VWF “activity” (VWF:Act). These tests seek to assess VWF-GPIb binding capacity independent of ristocetin, usually by using antibodies to the VWF A1 domain in ELISAs. These tests are easily confused with the VWF:RCo, which has also been referred to as “VWF activity.” None of these tests measures activity; rather both VWF:RCo and VWF:Act are sensitive to VWF conformation. The VWF:Act does not always provide the same results as VWF:RCo, particularly with regards to type 2M VWD, and is not considered a substitute for the VWF:RCo (reviewed in Ref. 290).

When type 2N VWD is suspected, VWF:FVIII binding capacity can be measured.²³⁴ Specific assays of FVIII binding to VWF (VWF:FVIIIB) have been developed and can be used to confirm the diagnosis of type 2N VWD.^300,301 Type 2N carriers do not always exhibit a decrease in VWF:FVIIIB, but a decreased VWF:FVIIIB-to-VWF:Ag ratio may correlate with heterozygosity for a type 2N VWF mutation.³⁰² Although this assay is widely used in European hemostasis laboratories, its availability in the United States is currently limited to a few specialized reference laboratories.

An assay measuring the VWFpp can be used to calculate the VWFpp:antigen ratio (VWFpp:Ag ratio) to detect a subset of VWD patients with decreased VWF survival. Good correlation has been reported between subjects with significantly shortened VWF half-life after DDAVP challenge and an increased VWFpp:Ag ratio.^287,303,304 This assay is currently available in a few reference laboratories. A normal platelet VWF:Ag in the setting of decreased plasma VWF laboratory parameters also suggests an accelerated clearance phenotype such as that seen in VWD type Vicenza,³⁰⁵ but platelet VWF:Ag testing also is not widely available in clinical laboratories.

A number of other assays for VWF activity have been developed. The PFA-100 system, which measures platelet binding under high shear,^306,307 is controversial in the diagnosis or monitoring of VWD. Although the PFA-100 is usually abnormal in type 2 VWD and in more severe type 1 and type 3 VWD cases, milder type 1 VWD and some type 2 VWD patients can have normal results.¹⁵⁰ Other VWF assays can measure binding of an antibody to the GPIb binding site on VWF as a proposed screening test for VWD.^{308,309,310,311} Additional assays can measure platelet agglutination induced by botrocetin (which is no longer commercially available) and other snake venom proteins.³¹² In the National Heart Lung and Blood Institute Expert Panel guidelines (http://www.nhlbi.nih.gov/files/docs/guidelines/vwd.pdf), none of these tests are recommended for screening for VWD.¹⁵⁰

With advances in understanding the molecular genetics of VWD, it is now possible to precisely diagnose and subclassify many variants of VWD on the basis of DNA mutations (reviewed in Ref. 313). DNA testing, particularly for type 2 VWD mutations which cluster within specific regions of the VWF gene (see Fig. 126-3), can be used to confirm the diagnosis and is available in specialized reference laboratories. The analysis of type 3 and type 1 VWD is more complex, as the currently known mutations are scattered throughout the gene²³⁵ and account only for a subset of patients.

The bleeding time is mentioned here for historical purposes only, as it was used as a screening test for VWD and other abnormalities of platelet function. Bleeding time varied considerably with the experience of the operator and a variety of other factors, did not prolong with FVIII deficiency, and correlated poorty with bleeding risk. Thus, the bleeding time is no longer recommended in the evaluation of VWD.¹⁵⁰

Given the mild clinical phenotype of most patients with the common variants of VWD, prenatal diagnosis for the purpose of deciding on terminating a pregnancy is rarely performed. However, type 3 VWD patients often have a profound bleeding disorder, similar to or more severe than classic hemophilia, and some families may request prenatal diagnosis. In those cases of VWD in which the precise mutation is known, DNA diagnosis can be performed rapidly and accurately by polymerase chain reaction (PCR) from amniotic fluid or chorionic villus biopsies (reviewed in Ref. 313) and would be expected to be compatible with new noninvasive prenatal testing methods.³¹⁴ In those cases where the mutation is unknown, diagnosis can still be attempted by genetic linkage analysis using the large panel of known polymorphisms within the VWF gene.³¹³ Although all cases of VWD analyzed to date appear to be linked to the VWF gene, the possibility of locus heterogeneity (i.e., a similar phenotype caused by a mutation in a gene other than VWF) should be considered.³¹⁵ As with all DNA testing, if prenatal testing is being considered, genetic counseling should be provided before the decision to test is made as well as following the procedure.

PLATELET-TYPE (PSEUDO-) VON WILLEBRAND DISEASE

Platelet-type (pseudo-) VWD is a platelet defect that phenotypically mimics VWD (Chap. 120). Patients have mucocutaneous bleeding, plasma VWF often lacks the largest multimers, RIPA is enhanced at low concentrations of ristocetin, and thrombocytopenia of variable degree is often present. Molecular analysis has identified missense mutations within the GPIbα gene as the molecular basis for pseudo-VWD. These mutations are located within the segment of GPIb that encodes the VWF binding domain and appear to induce the conformational change complementary to that produced in VWF by type 2B VWD mutations (reviewed in Ref. 316).

The specialized RIPA test should be performed at low ristocetin concentrations to distinguish type 2B and platelet type VWD from type 2A VWD. In this test, purified normal plasma VWF or cryoprecipitate added to platelet preparations from patients with platelet-type VWD causes platelet aggregation, distinguishing this disorder from type 2B VWD where patient platelets aggregate only at higher ristocetin concentrations. In addition, type 2B VWD plasma transfers the enhanced RIPA to normal platelets, whereas plasma from patients with platelet-type VWD interacts normally with control platelets.

ACQUIRED VON WILLEBRAND SYNDROME

Acquired VWD, or acquired von Willebrand syndrome (AVWS), is a relatively rare acquired bleeding disorder that usually presents as a late-onset bleeding diathesis in a patient with no prior bleeding history and a negative family history of bleeding (reviewed in Ref. 317). Decreased levels of FVIII, VWF:Ag, and VWF:RCo are common, and VWF multimers can be abnormal. AVWS is usually associated with another underlying disorder and has been reported to occur in patients with myeloproliferative neoplasms,³¹⁸ amyloidosis,³¹⁹ benign or malignant B-cell disorders,³²⁰ hypothyroidism,³²¹ autoimmune disorders,³²² several solid tumors (particularly Wilms tumor),³²³ cardiac or vascular defects (such as aortic stenosis),³²⁴ ventricular assist devices,³²⁵ or in association with several drugs, including ciprofloxacin and valproic acid.^326,327

The mechanisms that cause AVWS can generally be attributed to an associated medical condition. A variety of B-cell disorders have been associated with the development of anti-VWF autoantibodies. In most cases the AVWS appears to be due to rapid clearance of VWF induced by the circulating inhibitor, although these antibodies may also interfere with VWF function. Hypothyroidism results in decreased VWF synthesis.³²¹ In some cases of malignancy, AVWS is thought to be due to selective adsorption of VWF to the tumor cells or in myeloproliferative neoplasms, clearance/alterations of VWF by the high circulating platelet mass. AVWS associated with valvular heart disease, ventricular assist devices, or certain drugs, VWF may be lost by accelerated destruction or proteolysis under shear.^325,326,327

Although the VWF multimers in AVWS usually exhibit a type 2A pattern with relative depletion of the large multimer forms, AVWS can manifest as a wide range of VWD phenotypes.^322,328 Distinguishing AVWS from genetic VWD can be difficult, as testing for the associated autoantibodies is generally not available in the clinical setting. The diagnosis often rests on the late onset of the disease, the absence of a family history, and the identification of an associated underlying disorder.

Management of AVWS is generally aimed at treating the underlying disorder. VWF levels and bleeding symptoms often improve with successful treatment of hypothyroidism or an associated malignancy. Refractory patients have been treated with glucocorticoids, plasma exchange, intravenous gamma globulin, rituximab, DDAVP, and VWF-containing FVIII concentrates.^317,329

The mainstays of therapy for VWD are DDAVP, which induces secretion of both VWF and FVIII (reviewed in Ref. 330), and replacement therapy with VWF-containing plasma concentrates. The choice of treatment in any given patient depends upon the type and severity of VWD, the clinical setting, and the type of hemostatic challenge that must be met. Type 1 patients are most often treated with DDAVP alone, types 2A and 2B with a combination of DDAVP and a VWF-containing FVIII product, and type 2N and type 3 patients with VWF-containing concentrates.¹⁵⁰ A previous history of trauma or surgery and the success of previous treatment are important parameters to include in assessing the risk of bleeding. Prophylaxis is used in anticipation of hemostatic challenges,¹⁵⁰ such as dental extractions, and is efficacious in preventing recurrent bleeding in severe VWD patients.³³¹ Although in general there is a correlation between normal hemostasis and correction of VWF and FVIII activity, this does not occur in all cases.

DESMOPRESSIN

DDAVP is an analogue of antidiuretic hormone that acts through type 2 vasopressin receptors to induce secretion of FVIII and VWF, likely via cyclic adenosine monophosphate–mediated secretion from the Weibel-Palade bodies in endothelial cells.⁸³ When DDAVP is administered to healthy subjects, it causes sustained increases of FVIII and ristocetin cofactor activity for approximately 4 hours.³³² Patients with type 1 VWD treated with DDAVP release unusually high-molecular-weight VWF multimers into the circulation for 1 to 3 hours after the infusion.^332,333 Therapy with DDAVP often increases the FVIII activity, VWF:Ag, and ristocetin cofactor activity to two to five times the basal level.

DDAVP has become a mainstay for the treatment of mild hemophilia and VWD³³⁴ because it is relatively inexpensive, widely available, and avoids the risks of plasma-derived products. Approximately 80 percent of type 1 VWD patients have excellent responses to DDAVP, although this figure may be substantially lower depending on the criteria for diagnosis and response.³³⁵ It is regularly used in the setting of mild to moderate bleeding and for prophylaxis of patients undergoing surgical procedures. DDAVP is administered at a dose of 0.3mcg/kg continuous intravenous infusion over 30 minutes. DDAVP is also available for subcutaneous injection (at the same 0.3mcg/kg dose) and in intranasal form (at a fixed dose of 300mcg for adults and 150mcg for children), which appears to be similar in efficacy to intravenous administration,^336,337 although the response may be more variable.

The response to DDAVP in any given individual with VWD is generally reproducible and predicts response to future doses as long as the follow doses are at least 2 to 4 days later. In one study, 22 type 1 VWD patients showed a departure of less than 20 percent from the mean FVIII peak level calculated from two separate infusions. In addition, the consistency of response in one patient reliably predicted the future response of that patient and other affected family members.³³⁸ In a study of 77 type 1 VWD patients, DDAVP response was associated both with VWF mutation and baseline multimeric pattern, although subtle abnormalities in VWF multimers did not preclude a patient response to DDAVP. Interestingly, patients with the same VWF mutation did not necessarily exhibit the same degree of responsiveness to DDAVP, implying the influence of other factors in the magnitude of DDAVP effect.³³⁹ For patients requiring repeated infusions of DDAVP, the FVIII activity and VWF responses may not be of the same magnitude as after the first infusion. Although this decay in response has considerable individual variability, after one infusion of DDAVP per day for 4 days it was found that the responses on days 2 to 4 were reduced approximately 30 percent compared to day 1.^{336,337,338,340}

Therefore, in patients for whom DDAVP is potentially the treatment of choice, a test dose should be given at the planned therapeutic dose and route in advance of the first required course of treatment with measurements of before and after VWF and FVIII:C levels to ensure an adequate therapeutic response. Sampling additional time points after DDAVP infusion should be considered as some type 1 and type 2 VWD patients have a significantly shortened VWF half-life and it may be more appropriate to treat with VWF replacement therapy in clinical scenarios requiring more durable therapy to maintain hemostasis. For patients with type 1 VWD who are undergoing surgical procedures, DDAVP can be administered 1 hour before surgery and approximately every 12 hours thereafter for up to two to four doses before loss of clinically significant response. Patients should be monitored for response of FVIII and ristocetin cofactor activity and side effects, particularly hyponatremia (and then should be water restricted), when DDAVP is administered at frequent intervals. VWF-containing FVIII concentrates should be available for infusion as backup.

Approximately 20 to 25 percent of patients with VWD do not respond adequately to DDAVP. Type 2 VWD patients are less likely to have a response than type 1 patients,^341,335 and virtually no patients with type 3 VWD respond. The response to DDAVP of patients with type 2A VWD is variable. Although most patients respond only transiently, some patients exhibit complete hemostatic correction after DDAVP infusion.^342,343 It has been hypothesized that the differences in DDAVP efficacy among type 2A patients may correspond to the type of mutation, with better responses predicted in patients with group 2 mutations. A prospective study of the biologic response to DDAVP in well-characterized VWD patients included type 2A VWD patients with both group 1 and group 2 defects. Although patients with group 2 mutations had greater improvements in VWF:RCo and shortening of bleeding times than patients with group 1 defects, neither groups could be classified as responders.³⁴¹

Common side effects of DDAVP administration are mild cutaneous vasodilation resulting in a feeling of heat, facial flushing, tachycardia, tingling, and headaches. The potential for dilutional hyponatremia, especially in elderly and very young patients and with repeat dosing, requires appropriate attention to fluid restriction, as it may result in seizures. There have been isolated reports of acute arterial thrombosis associated with administration of DDAVP, but the risk appears to be very low when judged against the total number of patients treated. DDAVP is contraindicated in patients with unstable coronary artery disease because of increased risk of thrombotic events, such as myocardial infarction.³⁴⁴ Patients receiving DDAVP at closely spaced intervals of less than 24 to 48 hours can develop tachyphylaxis.³⁴⁰

Many experts consider DDAVP to be contraindicated in the treatment of type 2B VWD, as the high-molecular-weight VWF released from storage sites has an increased affinity for binding to GPIb and might be expected to induce spontaneous platelet aggregation and worsening thrombocytopenia.²²⁹ However, there are reports of DDAVP used successfully in type 2B VWD patients, with an associated shortening of bleeding times and variable thrombocytopenia.^345,346 Although type 2N patients can exhibit increased FVIII:C levels after DDAVP, in some cases the FVIII:C levels rapidly decline, likely a result of the absence of stabilizing normal VWF, attenuating clinical efficacy. Type 2M patients generally do not have a satisfactory response to DDAVP.^341,347

VON WILLEBRAND FACTOR REPLACEMENT THERAPY

It is important to determine the response to DDAVP for each individual so as to avoid the unnecessary use of plasma products. For type 3 VWD patients and other patients unresponsive to DDAVP, the use of selected virus-inactivated, VWF-containing FVIII concentrates is generally safe and effective.¹⁵⁰ Humate-P, Alphanate, Wilate, and Koate are all acceptable commercial VWF-containing plasma concentrates that have been evaluated in VWD replacement therapy in clinical studies, and other VWF-containing FVIII concentrates may also be effective. A plasma-free recombinant VWF–recombinant FVIII combination (rVWF-rFVIII) was recently evaluated in a phase III trial with severe VWD.³⁵⁵ All treated hemorrhagic episodes were rated as good or excellent (192 bleeds in 22 subjects). There were no adverse thrombotic events or allergic reactions. No antiVWF antibodies or anti-factor VIII antibodies were detected. In December 2015, the FDA-approved rVMF (Vonvendi) for on-demand treatment and to control bleeding episodes in adults (⋝18 years) diagnosed with VWD. The use of rVWF concentrate in the surgical setting is currently ongoing and may become available in the near future.³⁴⁸ Cryoprecipitate was useful in the past, but because it is not generally treated to inactivate bloodborne pathogens, is less targeted to correcting the VWD hemostatic defect, and its administration is associated with a large volume load, it is less desirable. It is important to note that most standard FVIII concentrates and all recombinant FVIII products are not effective in VWD because they lack clinically significant quantities of VWF. Although such products can substantially increase circulating FVIII:C, the infused factor is short-lived in the circulation in the absence of stabilizing VWF.³⁴⁹ Only preparations that contain large quantities of VWF with well-preserved multimer structure are suitable for use in VWD patients.

In practice, VWD replacement therapy dosing and timing has been largely empiric. Recommendations for therapy have been outlined based upon the degree and nature of hemorrhage and experience in clinical practice.¹⁵⁰ The objective is to elevate FVIII:C and VWF:RCo until bleeding stops and healing is complete. In general, replacement goals of FVIII:C and VWF:RCo should be initial replacement to greater than 100 IU/dL and maintenance of greater than 50 IU/dL for 7 to 14 days for major trauma, surgery, or central nervous system hemorrhage; greater than 30 to 50 IU/dL for 3 to 5 days for minor surgery or bleeding; greater than 50 IU/dL for delivery and continued for at least 3 to 5 days in the postpartum period; greater than 30 to 50 IU/dL for 1 to 5 days for dental extractions and minor surgery; and greater than 20 to 50 IU/dL for mucous membrane bleeding or menorrhagia. Laboratory monitoring of posttreatment FVIII:C and VWF levels is important in guiding therapy and avoidance of supratherapeutic replacement doses (>200 IU/dL VWF:RCo, >250 IU/dL FVIII), which are associated with an increased risk of thrombosis.^150,350,351 Although thrombosis is rare overall, VWD patients on prolonged therapy or with central access catheters appear to be at higher risk.³⁵²

In patients who have concomitant thrombocytopenia associated with or in addition to VWD, it may be necessary to transfuse platelets in addition to factor concentrates. If clinical bleeding continues, additional replacement therapy must be given and searches undertaken for other hemostatic defects. Type 3 VWD patients receiving multiple transfusions can develop antibodies directed against VWF (reviewed in Ref. 277). Continued replacement with VWF-containing concentrates is contraindicated because of the risk of anaphylaxis. A variety of approaches to the management of VWD inhibitors, similar to the treatment of FVIII inhibitors in hemophilia A (Chap. 123), have been attempted. Immunosuppression, recombinant FVIII, and recombinant factor VIIa have been reported to be useful in patients with type 3 VWD who have developed anti-VWF antibodies.

OTHER NONREPLACEMENT THERAPIES

Fibrinolytic inhibitors, such as ε-aminocaproic acid or tranexamic acid, have been used effectively in some VWD patients. Antifibrinolytics are commonly used alone or in conjunction with DDAVP or a plasma-derived VWF replacement product in patients with gynecologic bleeding, mucous membrane bleeding, or undergoing dental procedures.¹⁵⁰ Fibrinolytic inhibitors can be delivered systemically or topically and are generally well tolerated, but rarely can cause nausea or diarrhea and are contraindicated in patients with gross hematuria.

Estrogens or oral contraceptives have been used empirically in treating menorrhagia. In addition to their effects on the ovaries and uterus, some estrogens can increase plasma VWF levels. Patients with VWD frequently normalize their levels of FVIII, VWF:Ag, and VWF:RCo during pregnancy (Chap. 8). Postpartum hemorrhage within the first few days after parturition may be related to the relatively rapid return of FVIII and VWF activities to prepregnancy levels, and postpartum hemorrhage in all forms of VWD may occur as long as 1 month postpartum. In pregnant patients with type 1 VWD, the FVIII and ristocetin cofactor activities usually rise above 50 percent. These patients usually do not require any specific therapy at the time of parturition. In contrast, individuals who have 30 percent or less FVIII or variant forms of VWD are more likely to require prophylactic therapy before delivery. In a recent study, women receiving treatment for VWD postpartum were unexpectedly found not to have corrected to target levels.³⁵³ Therefore, laboratory testing is recommended at term and should be considered in the postpartum period in patients at risk for immediate and/or delayed bleeding complications or receiving therapy.

Recombinant activated factor VII (rFVIIa, or NovoSeven) has also been successfully used in VWD patients with severe hemorrhage refractory to VWF replacement therapy and in bleeding patients with anti-VWF antibodies (reviewed in Ref. 354). In the case of minor accessible bleeding, topical drugs such as fibrin sealants or topical bovine thrombin may also be considered when standard VWD therapies fail to provide adequate local hemostasis.¹⁵⁰