How we make an accurate diagnosis of von Willebrand disease

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Review Article

How we make an accurate diagnosis of von Willebrand disease ⁎

Luciano Baronciania, , Flora Peyvandia,b a b

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, Milan, Italy Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy

A R T I C LE I N FO

A B S T R A C T

Keywords: von Willerand factor von Willebrand disease Diagnosis Chronic disease Acute bleeding Disease burden

von Willebrand disease (VWD) is a common autosomally inherited hemorrhagic disorder mainly associated with mucocutaneous bleeding. VWD is due to quantitative (type 1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF), a large multimeric plasma glycoprotein that plays a relevant role in hemostasis. VWF is essential to mediate platelet adhesion and aggregation at the sites of vascular injury under high shear stress conditions. VWF also carries coagulation factor VIII (FVIII), prolonging its half-life and concentrating it at the site of the damaged endothelium. The diagnosis of VWD, in agreement with the International Society for Thrombosis and Hemostasis guidelines, requires several assays that are necessary to evaluate the capacity of VWF to interact with several ligands, e.g. platelet glycoprotein Ibα, collagen and FVIII. Therefore, the differential diagnosis of VWD patients as type 1, 2A, 2B, 2M, 2N or 3 is a prerogative of specialized laboratories, where specific tests, like multimer analysis or ristocetin-induced platelet agglutination, are performed routinely. On the other hand, the basic identification of patients with VWD is nowadays possible in many hemostasis laboratories thanks to the availability of automated tests that measure in patient plasma VWF antigen levels and its plateletdependent activity. Nevertheless the laboratory investigation for VWD of a subject referred for a hemorrhagic tendency should start only after the attending physician, after evaluation of his/her personal and family bleeding history, confirmed the suspicion for VWD. The purpose of this manuscript is to give an overview of the complex process that leads to the diagnosis of the VWD.

1. Introduction von Willebrand disease (VWD) is a common inherited bleeding disorder associated to defective platelet adhesion/aggregation and often reduced factor (F)VIII plasma levels. It is inherited with a dominant or recessive autosomal pattern and therefore affects both men and women, although especially in its milder form women are more often symptomatic. It is due to the deficiency, dysfunction or both of von Willebrand factor (VWF), a multimeric large adhesive plasma glycoprotein that plays a major role in primary hemostasis. In the last ten years many functions have been recognized to VWF [1], but in primary hemostasis its main role is the ability to mediate platelet adhesion and aggregation at sites of vascular injury. Due to its large size, VWF is capable to interact with exposed subendothelial collagen fibrils and circulating platelets, mediating their adhesion and aggregation. Under high blood shear rate conditions, as in capillaries and arterioles, VWF is essential to support platelet adhesion, because platelet collagen receptors only mediate platelet adhesion at low blood shear rates [2]. VWF has also a role in secondary hemostasis by circulating in plasma as

a complex with coagulation FVIII, protecting it from premature cleavage and thus prolonging its half-life [3]. Therefore, deficiency of VWF may secondarily impair blood coagulation. VWD patients show heterogeneous bleeding symptoms from mild to severe, broadly reflecting the severity of VWF deficiency/dysfunction. Hemorrhagic manifestations are mainly involving mucocutaneous tracts, due to the important role that VWF plays under high blood shear rates in small blood vessels. Different assays are needed to evaluate quantitative and qualitative defects of VWF, and on the basis of the results of these laboratory tests different types of VWD can be identified. Several steps are necessary in order to reach a diagnosis of VWD in a patient reporting hemorrhagic manifestations: 1) the evaluation of patient personal and family bleeding history, 2) the hemostasis screening tests, 3) the first-level tests for VWD screening, 4) the second-level tests for VWD typing and 5) the molecular analysis. The latter step is indeed not essential to obtain the patient diagnosis if all biochemical assays are available, but in some instances it may represent an important confirmation of the biochemical diagnosis. The purpose of this manuscript is to give an overview of the complex process that leads to the diagnosis of the VWD

⁎ Corresponding author at: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, via Pace 9, 20122 Milan, Italy. E-mail address: [email protected] (L. Baronciani).

https://doi.org/10.1016/j.thromres.2019.07.010 Received 1 April 2019; Received in revised form 27 June 2019; Accepted 14 July 2019 0049-3848/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Luciano Baronciani and Flora Peyvandi, Thrombosis Research, https://doi.org/10.1016/j.thromres.2019.07.010

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Fig. 1. Panel (A) features the schematic structure of the precursor of VWF (pre-pro-VWF) with the homologous repeated domains. Locations of intersubunit disulfide bonds involved in dimerizatiom and multimerization are shown together with the binding sites for several ligands. Arrows point out the position of von Willebrand disease (VWD) type 2 mutations. Recessive VWD type 2 variants are indicated by an *. Panel (B) shows the multimeric pattern obtained using a low resolution gel [69] of the type 2 VWD variants in comparison with normal plasma (NP) run in parallel. The dark arrows show the location of mutations that correspond to type 2A variants with the characteristic multimeric pattern obtained by an intermediate resolution agarose gel (Pannel C). The “close-up” in the square shows the bands of the “triplet structure” as it appears in NP, a central heavier marked band surrounded by two lighter satellite bands, one above and one below. Type 2A (IIA) shows markedly increased triplet bands caused by mutations in the A2 domain that result in an increased proteolysis by ADAMTS13. On the other hand, type 2A (IIC) and (IIE) show the absence of the triplet satellite bands as a consequence of a defective multimer assembly due to mutations in the propeptide (D1 and D2 domains) or in the D3 domain, respectively. The absence of physiologic large VWF multimers in these variants results into a reduced proteolysis by ADAMTS13 [67]. Type 2A (IID) is characterized by defective multimer assembly caused by mutations in the cysteine knot domain and is characterized by the presence of “odd” band size in the multimer analysis using intermediate resolution gel [68]. In this figure the type 2A(IID) variant has not been reported using the low resolution analysis gel because this variant was never identified in our laboratory, and so the intermediate resolution gel (agarose 1.7%) of the type 2A(IID) has been kindly provided by Prof. Ulrich Budde. VWF, von Willebrand factor; FVIII, factor VIII; GPIbα, glycoprotein Ibα; GPIIb/IIIa, glycoprotein IIb/IIIa; ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13; HMWM, high molecular weight multimers; NP, normal plasma; CK, cysteine knot domain.

VWFpp, the D′ and D3 domains of the dimers, low pH and high Ca++ concentrations. Under these conditions VWF multimers are formed and further assembled into a tubular conformation before secretion or storage inside the Weibel-Palade bodies of the endothelial cells or platelet α-granules. The size of the VWF multimers stored in these organelles exceeds the size of multimers circulating in plasma and is therefore called ultra-large VWF (ULVWF). After their release into the circulation the ULVWF remain temporarily attached to the cell surface, uncoiling themselves and allowing the plasma protease ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13) to cleave them and so reduce their size [8].

or to its exclusion. 2. von Willebrand factor VWF is synthesized in endothelial cells and megakaryocytes as a pre-pro-VWF of 2813 amino acids (22-residue signal peptide, 741-residue propeptide and 2050-residue mature subunit). More than 95% of the pro-VWF sequence refers to structural domains as follows: D1-D2D′-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK [4,5]. Some of these domains interact specifically with the VWF ligands, others are responsible for the protein biosynthesis as shown in Fig. 1. VWF biosynthesis is a complicated process, that includes several post-translational modifications of the newly synthesized pre-pro-VWF before becoming the large hemostatically active glycoprotein known as VWF [6]. The signal peptide is removed immediately in the endoplasmic reticulum (ER), where pro-VWF undergoes a glycosylation process. Dimerization also occurs in the ER by disulphide bonding between the cysteine residues in the cystine knot (CK) of the pro-VWF. Polymerization of pro-VWF dimers (multimerization) occurs in the Golgi apparatus, along with a further glycosylation process. The VWF propeptide (VWFpp) is cleaved but remains noncovalently bound to the dimers and mediates the polymerization process that results in the formation of VWF multimers [7]. This process requires the presence of the D1 and D2 domains of the

3. Function of von Willebrand factor in primary hemostasis VWF is sensitive to high shear stress conditions, which modify the protein folded conformation into an unfolded active conformation [6]. Therefore, under conditions of physiologic blood flow VWF coexists without major interactions with platelets. At variance, VWF interacts spontaneously with subendothelial collagen fibrils even at a low shear stress conditions. At sites of vascular injury, VWF mediates platelet binding to the exposed collagen fibrils of the subendothelium and then promotes aggregation resulting in formation of the platelet plug. The role of platelets in hemostasis is to become irreversibly attached to the 2

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damaged vessel wall. The first physical factor that impairs platelet binding to the vessel wall is blood flow velocity, that is higher at the center of the vessel and lower close to the vessel wall. These changes in velocity amid layers of fluid result in a shearing effect, or shear stress. Damage to the vascular endothelial surface causes in the exposure of the subendothelium matrix and a modification of the rate of blood flow, resulting in an increased shear stress. The VWF folded conformation is shear-stress sensitive and under the increment of shear forces VWF unravels itself exposing all its binding sites [6]. Plasma VWF binds to the exposed collagen fibrils with high affinity and then the immobilized VWF interacts with the platelet glycoprotein (GP) Ibα receptor through its A1 domain, tethering platelets at the site of damaged endothelium (Fig. 1). However, due to the fast dissociation rate of this interaction, platelets tethered to the vessel wall keep moving in the direction of the blood flow, but at slower speed. The activation of the platelet integrin GPIIb-IIIa is required in order to obtain firm platelet adhesion, this integrin being responsible for platelet-to-platelet interactions which are mediated by VWF and, at low flow condition, fibrinogen. Due to its slow rate of binding to VWF the GPIIb-IIIa does not contribute to the early events of platelet adhesion. However, after platelet activation, due to the continuous VWF-GPIbα catch and release interactions GPIIb-IIIa modifies its conformation and then increases its affinity for the VWF C4 domain (Fig. 1). This change, along with the reduced motion of platelets, favors the formation of a stable bond between the VWF C4 domain and the GPIIb-IIIa integrin, thus binding platelets irreversibly to the exposed subendothelium [9].

Table 1 Classification of von Willebrand disease [12,15]. Type

Definitions

Low VWF 1 2 2A

Partial reduce VWF levels (> 30 IU/dL) Partial quantitative deficiency of VWF Qualitative deficiency of VWF Decreased platelet-dependent VWF adhesion due to selective deficiency of high-molecular-weight VWF multimers Increased affinity of VWF for platelet glycoprotein Ibα Decreased platelet-dependent VWF adhesion with normal multimeric pattern Markedly decreased VWF binding affinity for factor VIII Virtually complete deficiency of VWF

2B 2M 2N 3

VWF, von Willebrand factor.

and laboratory features of platelet-type VWD are almost identical to those of VWD type 2B and therefore a specific laboratory approach is necessary to distinguish these different diseases. A specific test is also required to do the differential diagnosis between mild forms of hemophilia A and VWD type 2N. Hemorrhagic manifestations similar to those associated with VWD may arise in older subjects with no family history and a recent bleeding history. The acquired von Willebrand syndrome (AVWS) is due to a deficiency or dysfunction of plasma VWF usually associated with a heterogeneous group of clinical conditions, like cardiovascular, immunological, myeloproliferative and lymphoproliferative diseases and in solid tumours [17]. The laboratory assays used for patients with inherited disease can be exploited to establish the severity of the acquired disease and monitoring therapy [18].

4. Function of von Willebrand factor in secondary hemostasis Von Willebrand factor and FVIII circulate in plasma as a macromolecular. The interaction between VWF and FVIII is mediated by the amino acid residues from 764 to 1036 in the D' and D3 domains (Fig. 1) [10]. This association causes the prolongation of the FVIII half-life by preventing its premature cleavage by a variety of serine proteases, such as for instance activated protein C [3]. In addition, binding of FVIII to VWF has also the purpose of delivering FVIII at the sites of vascular injury, thus increasing its low plasma concentration (1 nM) where it is mostly needed [11].

6. Evaluation of family and personal bleeding history Hemostasis screening tests and VWD first level assays should always follow the physician evaluations of the patient bleeding history. Thus, the evaluation of patients hemorrhagic symptoms, along with the information collected on patient family, represent the first step in the diagnostic process and also the most crucial one, because it may prevent further evaluation of the patient. The nature of bleeding symptoms, their frequency, the timing of bleeding after trauma, the age of the first hemorrhagic manifestation and the presence of a family history of bleeding disorders are useful to support or exclude the suspicion of VWD. The manifestations of various bleeding symptoms suggest a possible hemostatic defect, whereas repetitive bleeding episodes at the same site (e.g. unilateral epistaxis or only menorrhagia) are more likely to be caused by local issues. The type of bleeding symptoms plays a relevant role in suggesting the possible hemostatic defect. In VWD, the hemorrhagic manifestations mainly regard mucocutaneous tissues, due to the essential role that VWF plays under high blood shear rates that occur in small blood vessels. Therefore, the main mucocutaneous bleeding symptoms are epistaxis, gingival bleeding, menorrhagia and gastrointestinal bleeding. In addition, procedures like dental extractions and surgery and hemostasis challenging events such as childbirth or major trauma are responsible for bleeding in VWD. Due to the different deficiency or dysfunction of VWF symptoms can be quite different from patient to patient, with individuals who exhibit bleeding only after major surgery and others, more severely affected, who present severe spontaneous bleeding as hemarthroses and soft-tissue hematomas. An evaluation of the severity of the hemorrhagic disease can be done by establishing at which age the first bleeding manifestations appeared and evaluating the frequency of the bleeding episodes. In order to do so, specific questionnaires have been proposed with quantitative scoring systems, with the goal to estimate the severity of the disease [19,20]. Tosetto et al. proposed in 2006 a bleeding score validated in the frame of the European study on VWD type 1 and it is probably the most used worldwide [20]. The Standardization and Scientific Committee of the International Society for Thrombosis and Hemostasis (ISTH) had proposed a Bleeding Assessment Tool (BAT)

5. von Willebrand disease Even though von Willebrand disease (VWD) is often reported as the most common inherited bleeding disorder, accurate data concerning its prevalence are still not available in different regions of the world. Population studies reported a prevalence from 0.6 to 1.3% [12,13] but surveys of symptomatic individuals in specialized hemostasis centers reported rates of 0.01% or lower [14]. The most common symptoms of mild VWD are mucosal bleeding (epistaxis, gingival bleeding, menorrhagia) and prolonged bleeding after surgical procedures and dental extractions. Hemarthroses and soft-tissue hematomas occur frequently only in severely affected individuals (VWD type 3) who are also partially FVIII deficient. VWD classification is based on the quantitative (type 1 and 3) or qualitative (type 2) defects of VWF (Table 1) [15]. VWD type 1 presents with a proportional decrease of VWF antigen and activity, a normal or nearly normal multimeric pattern and is usually inherited with a dominant pattern. VWD type 3 is characterized by a virtually absent (undetectable) VWF in both plasma and platelets. It is the most severe form of the disease and is always inherited with a recessive pattern. VWD type 2 is the most heterogeneous form of VWD, characterized by a disproportion of VWF activity to the VWF antigen level. VWD type 2 is further divided in type 2A, 2B, 2M and 2N on the basis of the dysfunctional activities of VWF (Table 1). Most of the type 2 forms are inherited in the dominant pattern but in type 2N and in some type 2A cases inheritance is recessive. Platelet-type VWD, also known as pseudo-VWD, is a platelet disorder characterized by increased affinity of the platelet GPIbα receptor for the VWF A1 domain [16]. The clinical 3

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be present [26]. In many laboratories the evaluation of the BT has been replaced by the PFA 100 [27,28]. The PFA 100 closure time is often prolonged in severe platelet function disorders and severe VWD [29,30]. However, we usually do not perform the PFA 100, because the test is not able to detect all VWD forms [31] and therefore we prefer to rely directly upon the first level tests (see below). The screening for secondary hemostasis defects can be performed by using the prothrombin time (PT) and the activated partial thromboplastin time (APTT). The PT is normal in VWD but the APTT may be prolonged, due to the reduced level of FVIII that might be present. Nevertheless, this test is often not informative for diagnosis, because of normal FVIII levels in the commonest mild forms of VWD, in type 2 variants and also due to the possible compensatory effect of other coagulation factors involved in the APTT test (i.e. factor (F)IX, FXI and FXII). For these reasons the evaluation of the first level tests should be performed regardless of the results obtained with the hemostasis screening tests. Table 2 shows both the first and second levels tests for VWD diagnosis. Using the former we can confirm the presence of the disease, by using the latter we are able to characterize the VWF variants and therefore to identify the different VWD types.

available at the web site (https://www.isth.org/page/reference_tools; last visit June 2019). BATs are very useful to compare the bleeding severity between different types of VWD patients or between cohorts of patients. However, as diagnostic tool BATs have limitations, because the corresponding scores are age dependent and the lack of hemostasis challenges in children make the use of BATs difficult. Moreover, the frequency of bleeding symptoms is not reported and symptoms are not disease specific. The use of BATs is also time demanding and thus is not practical in every-day clinical activities [21,22]. Nevertheless, BATs have high negative predictive values in screening individuals for bleeding disorders and therefore are useful to exclude the presence of a hemorrhagic disease [23]. In addition to the patient personal bleeding history the identification of other family members presenting with a bleeding history is quite relevant in supporting the suspicions of VWD. The relevance of family bleeding history has been underlined by Tosetto et al.[24] who evaluated the different contributions of the “bleeding symptoms”, “reduced VWF level” and the “inheritance of the disease” for the diagnosis of type 1, the most common form of VWD but also the most challenging to be diagnosed. Their study shows that the most significant clue to diagnosis is the inheritance of the phenotype (considered as the number of first-degree family members with reduced VWF levels), followed by reduced VWF levels and bleeding symptoms in the proband. Therefore, the presence of other family members (one first-degree relative or two second-degree relatives) with personal history of significant mucocutaneous bleeding and laboratory test compatible with VWD type 1 [25] markedly increases the odds of the proband to have VWD. The lifelong evaluation of personal and family history of bleeding is an important tool to distinguish between patients affected with inherited VWD and those with the AVWS, because this distinction may be difficult when relying only on laboratory assays.

8. First level tests for VWD diagnosis First level tests include the measurement of plasma FVIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag) and platelet-dependent VWF activity, frequently evaluated by mean of the ristocetin cofactor activity (VWF:RCo) assay. The normal range is generally between 50 and 150% or IU/dL for FVIII:C, VWF:Ag and VWF:RCo. Even though the guidelines do not require this, it may be appropriate to have two different ranges, one using normal subjects with blood group O and another using normal subjects with blood group noneO, because individuals with blood group O have 25% lower plasma levels of VWF than subjects with blood group non-O [32]. The identification of the patient blood group is usually done along with the first level assays. Another aspect that should be taken into consideration is the age of the patient, because VWF levels in the general population increase with ageing [33]. Also stress or higher hormone levels can affect the amount of VWF in plasma. In children who are crying or struggling during a blood withdrawal VWF levels may increase and could mask the presence of a mild form of VWD, and anxiety could do the same in adults or hormonal therapy in women. Guidelines published from the National Heart, Lung, and Blood Institute Expert Panel report (USA) suggest for the diagnosis of type 1 that the VWF:Ag or VWF:RCo plasma levels are < 30 IU/dL. They also suggest that individuals with VWF:Ag levels between 30 and 50 IU/dL should be reported as “low VWF” [12]. This suggestion is due to the fact that some studies in large VWD type 1 cohorts [34,35] demonstrated that the association between hemorrhagic symptoms and the presence of a VWF gene mutation was high only in patients with VWF levels < 30 IU/dL, but not in those with levels between 30 and 50 IU/dL. This might be explained by the knowledge that mild hemorrhagic manifestations are common in the normal population and similarly common are the individuals with borderline levels of VWF, so that these two situations may be found together only accidentally. Subjects with “low VWF” [12] should not be considered ill but only exposed to a higher risk of bleeding [36].

7. Hemostasis screening tests Hemostasis screening tests useful to diagnose VWD are in Table 2. The screening for primary hemostasis defects can be done assessing the platelet count, patient skin bleeding time (BT) or the Platelet Function Analyzer (PFA 100) closing time. Special attention should be given to the platelet count, because in type 2B a mild thrombocytopenia might Table 2 Description of the steps necessary to diagnose VWD patients. Step 1- Evaluation of patient personal and family bleeding history Bleeding assessment tool Step 2 - Hemostasis screening tests Evaluation of primary hemostasis Platelet count Evaluation of secondary hemostasis (COAGULATION) Prothrombin time (extrinsic pathway) Activated partial thromboplastin time (intrinsic pathway) Step 3 - VWD classification 1st level tests VWF:Ag Platelet-dependent VWF activity (VWF:RCo, VWF:Ab, VWF:GPIbR or VWF:GPIbM) FVIII:C Step 4 - VWD classification 2nd level tests VWF multimer analysis VWF:CB VWF:FVIIIB VWFpp RIPA Intraplatelet VWF Step 5 - Molecular analysis of VWF gene

8.1. FVIII coagulant activity (FVIII:C) The measurement of FVIII:C can be obtained using the one-stage clotting assay or the chromogenic assay, both being reliable methods to measure FVIII:C in normal individuals and in patients with VWD [37]. Type 3 patients may have values of FVIII:C lower than 10 IU/dL, whereas in type 1 and type 2A, 2B and 2 M FVIII:C levels may be decreased to a variable extent but are often normal. In type 2N VWF levels are normal, but the reduced capacity of this variant to bind FVIII impairs FVIII survival in the circulation, resulting in low FVIII:C levels.

VWF:Ag, VWF antigen; VWF:RCo, VWF ristocetin cofactor activity: assays that use platelets and ristocetin; VWF:Ab, assays that are based on the binding of a monoclonal antibody to a VWF A1 domain epitope; VWF:GPIbR, assays that use ristocetin-induced binding of VWF to a recombinant wild-type GPIb fragment; VWF:GPIbM, assays based on the spontaneous binding of VWF to a gain-offunction mutant GPIb fragment [46]; FVIII:C, factor VIII coagulant activity; VWF:CB, VWF collagen binding; VWFpp, propeptide of VWF; VWF:FVIIIB, VWF binding to FVIII assay, RIPA, ristocetin-induced platelet agglutination assay. 4

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8.4. The platelet-dependent VWF activity/VWF:Ag and FVIII:C/VWF:Ag ratios

8.2. VWF antigen concentration (VWF:Ag) The measurement of the VWF:Ag in patient plasma can be done with several assays. Typically evaluated with an enzyme-linked immunosorbent assay (ELISA) [38,39], in many laboratories VWF:Ag is now often measured using an automated latex immunoassay (LIA) [40] or an automated chemiluminescent assay [41]. The chemiluminescent and the ELISA assays have limits of detection of 0.5 and 1 IU/dL, lower than that of the LIA assays (limit of detection of 10 IU/dL). Therefore, in the cases with severe quantitative VWF defects, the latter methods are not suitable to discriminate between type 3 (VWF:Ag < 1 IU/dL) or severe type 1 (VWF:Ag < 5 IU/dL) [15].

In normal subjects and in VWD type 1 the level of platelet-dependent VWF activity reflects the concentration of VWF:Ag and the platelet-dependent VWF activity/VWF:Ag ratio is always higher than 0.6. However, in patients with type 2 platelet-dependent VWF activity can be severely decreased (ratio < 0.6) owing to the absence of the high molecular weight multimers (HMWM), as in type 2A or 2B or owing to the presence of mutations in the A1 domain that reduce its affinity for the GPIbα, as in type 2M or, more rarely, due to both situations [59,60]. Therefore, the platelet-dependent VWF activity/VWF:Ag ratio is able to differentiate most type 2 patients from those with type 1. However, this is not true for cases with type 2M with a collagen binding defects [61], in those of type 2B with normal HMWM (e.g. type 2B New York) [62,63] and in type 2N. In the latter the alternative FVIII:C/VWF:Ag ratio is useful, beacuse this ratio is usually > 0.6 in normal subjects and in all VWD patients except those with type 2N, who have a FVIII:C/ VWF:Ag < 0.6 as in hemophilia A. An increase of the FVIII:C/VWF:Ag ratio (> 2) has also been reported in heterozygous carriers of VWD type 3 defects, so that the identification of high ratio in a patient suggests a reduced synthesis or secretion of VWF [64].

8.3. Platelet-dependent VWF activity This activity, that reflects the capacity of VWF to interact with the platelet GPIbα receptor, is historically assessed using the ristocetin cofactor (VWF:RCo) assay. This method measures the capacity of VWF to agglutinate normal exogenous platelets (formaldehyde-fixed or lyophilized) in the presence as a cofactor of the antibiotic ristocetin, using either an aggregometer or a coagulometer [42–44]. VWF in the absence of high shear forces does not bind to its platelet GPIbα receptor, but the added ristocetin binds to VWF and promotes its capacity to interact with platelets [6]. The VWF:RCo activity is important for VWD typing and also for monitoring patient replacement therapy, at least in some clinical situations such as mucosal tract bleeding episodes. However, these assays evaluate the platelet-dependent VWF activity under nonphysiological conditions and present several limitations, as a high assay variability and poor sensitivity at low plasma levels (VWF:Ag < 10 IU/ dL) [45]. In the last decade, several new assays were introduced to evaluate the platelet-dependent VWF activity in order to overtake some of the VWF:RCo assays weaknesses and a new nomenclature was introduced in order to distinguish these new assays [46]. First platelets have been replaced with the more stable latex particles covered with a monoclonal antibody specific for an epitope of the A1 domain involved in VWF binding to the GPIbα platelet receptor in the frame of an immunoturbidimetric assay that did not require ristocetin (VWF:Ab) [47,48]. Subsequently, platelets have been replaced with latex particles or magnetic beads covered with recombinant (r)GPIbα and used along with ristocetin (VWF:GPIbR) [48–52]. In this new method the use of latex particles covered with mutant gain-of-function rGPIbα molecules that spontaneously bind VWF allowed to exclude ristocetin (VWF:GPIbM) [51–56]. Several studies have shown that the automated VWF:Ab, VWF:GPIbR and VWF:GPIbM assays perform well and yield results close to the reference method VWF:RCo [41,48–57]. However, Boender et al. [58] have recently reported the evaluation of plateletdependent VWF activity in 661 VWD patients using four different methods (VWF:RCo, VWF:Ab, VWF:GPIbR and VWF:GPIbM). This is the largest group of patients evaluated in these comparative studies and showed that although the four methods correlated well one with another (R2 > 0.90) one-fifth of the patients showed clinically relevant differences. In comparison with the VWF:RCo, all the other tests have shown higher sensitivity and superior diagnostic accuracy. The new assays also offer more stable reagents, which allowed the test to be performed on demand even for few samples without increasing its cost. This advantage, along with those mentioned above, contributed to the replacement of the VWF:RCo assay with VWF:GPIbR and VWF:GPIbM assays in many laboratories. Therefore, to avoid confusion in this manuscript we shall not use any abbreviation to refer to the plateletdependent VWF activity but we report it in full, except when we are referring specifically to one of the four assays (VWF:RCo, VWF:Ab, VWF:GPIbR or VWF:GPIbM).

9. Second level tests for VWD diagnosis 9.1. VWF multimer analysis This assay, that evaluates the VWF molecules with different molecular weights is performed using electrophoresis on an agarose/sodium dodecylsulfate (SDS) gel run under non reducing conditions. VWF circulates in plasma as a population of molecules of different size from 540 kDa up to 20,000 kDa. Therefore in this electrophoretic analysis VWF is visualized as several bands of different molecular weight. The faster moving bands are conventionally indicated as low molecular weight multimers, followed by the intermediate molecular weight multimers, whereas the slower moving bands are indicated as high molecular weight multimers (HMWM) (Fig. 1) [65]. The HMWM are the most effective in mediating platelet adhesion and the absence or decrease of these bands is associated to VWD type 2A or 2B. Electrophoreis gels at low resolution (e.g. 1.2% HGT agarose/ 0.1% SDS) are used to evaluate the defect of the HMWM, but using gel at an intermediate resolution (e.g. 1.6% LGT agarose/0.1% SDS) it is possible to visualize the inner structure of each VWF band or oligomer, so that every band is resolved in at least three bands, known as the triplet structure (Fig. 1). The triplet presents a central heavier marked band surrounded by two lighter bands, one above and one below, the lighter bands being produced by the physiologic cleavage of VWF by the plasma protease ADAMTS13. The multimer analysis at intermediate resolution can be used to evaluate the different mechanisms responsible for VWD type 2A (IIA, IIC, IID and IIE) (Fig. 1) [66–68]. The preparation of the agarose gel used for the multimer analysis and its handling can be a difficult process to reproduce with a good quality standard. Therefore, only specialized laboratories usually set up this technique, but a new commercial method that uses pre-casted gels, has become recently available to perform the multimer analysis at low resolution [69]. The kit uses electrophoretic instruments that are common in many biochemical laboratories and offers the additional possibility to obtain a quantitative evaluation with the percentage of the HMWM versus the total VWF multimer pattern. 9.2. VWF collagen binding (VWF:CB) The VWF:CB is mainly evaluated using ELISA methods [39], although an automated chemiluminescent method is also available [70]. Generally the collagen used in these assays is of type I, type III or a mix of the two. The VWF:CB assay is sensitive to detect the absence of 5

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agglutination is obtained with amounts of ristocetin of 0.7 mg/mL or smaller, as in the case of type 2B or platelet-type VWD patients [89]. On the contrary, RIPA is decreased when the concentration of ristocetin needed to obtain platelet agglutination is above 1.2 mg/mL, as in type 2A and 2M. There is a relationship between the levels of platelet-dependent VWF activity and the amount of ristocetin necessary to induce agglutination in RIPA. In the normal population, subjects with 150 IU/ dL of platelet-dependent VWF activity require low amounts of ristocetin (i.e. 0.8 mg/mL) to obtain 30% of platelet agglutination, whereas subjects with 50 IU/dL of platelet-dependent VWF activity will require higher amount (1.2 mg/mL). The relation between these two assays should always be considered in the evaluation of VWD patients. An extreme example is our case of a suspected type 2A patient with 7 IU/dL of VWF:RCo and 1.2 mg/mL of RIPA, who did turn out to be a compound heterozygote for a type 2B variant and a type 1 defect [90]. In several occasions patients with severe type 2B mutations (p.Arg1306Trp and p.Arg1308Cys), that cause the loss of HMWM and intermediate MWM, did show a RIPA value within the normal range (0.8 and 0.9 mg/mL). However, the same patients showed VWF:RCo to be < 6 IU/dL and therefore the RIPA of 0.9 mg/mL could be explained only by the presence of a type 2B mutation [26]. The RIPA assay is influenced by VWF:Ag levels, the absence of the HMWM, the presence of mutations in the A1 domain (type 2B and 2M) and in the GPIbα receptor (platelet-type VWD). For these reasons RIPA can be used to differentiate type 2B from type 2A and 2M, whereas it is more difficult to distinguish with RIPA type 2B and platelet-type VWD. This distinction can be done, in the case of an enhanced RIPA, by repeating the assay using patient plasma with the platelets of a normal control and vice versa. This could help to understand whether the enhancement is associated to the patient plasma or to the patient platelets. However, due to the “sticky” nature of platelets, the performance of these mixing tests is not so simple and a valid and easier alternative is the use of molecular biology to identify the gain-of-function mutations in either the VWF or in the GP1BA genes [91].

HMWM and therefore it has been proposed as a surrogate of the multimer analysis. Indeed, it can be useful to differentiate type 2A and 2B from type 2M. The use of VWF:CB with collagen type I and III has also allowed the identification of mutations in the A3 domain that present with a reduced capacity of VWF to bind collagen despite the presence of a normal multimeric pattern (VWD type 2M) [61,71]. Collagens of type IV and VI have been also used by some investigators, allowing the identification of some mutations in the A1 domain [72–74]. 9.3. VWF binding to FVIII (VWF:FVIIIB) The evaluation of the VWF:FVIIIB is performed using an ELISA assay that evaluates the capacity of VWF in the test plasma to bind an exogenous source of recombinant FVIII. Using this assay it is possible to discriminate between a mild form of hemophilia A, which is X-linked inherited, and VWD type 2N, inherited with an autosomal recessive pattern [75]. The assay is also able to identify the asymptomatic carriers of type 2N. Similar findings may also materialize owing to mutations in the D3 domain, that are known to impair the VWF multimerization process. In some of these patients the VWF:FVIIIB is mildly reduced and the FVIII:C levels are proportional to the VWF:Ag values [76]. VWF:FVIIIB is affected only by the presence of mutations in the D′ and D3 domains. However, in some acquired forms secondary to lymphoproliferative diseases, we found a mildly reduced VWF:FVIIIB, probably due to the presence of anti-VWF antibodies. 9.4. VWF propeptide (VWFpp) The VWFpp (amino acid residues 23-763 of the pre-pro-VWF) is synthesized at the same molar ratio of the mature subunit of VWF (amino acid residues 764-2813). However, most VWFpp molecules circulate in plasma independently from VWF and the different half-lives of the VWFpp and VWF, i.e. 2–3 h and 8–14 h respectively [77,78], generate a VWFpp/VWF:Ag ratio that in the normal population ranges from 0.6 to 1.6 [79]. The VWFpp can be measured by ELISA with antihuman VWFpp antibodies [80] and its measurement, particularly the VWFpp/VWF:Ag ratio, can be used to estimate the rate of clearance of plasma VWF, assuming that the clearance rate of the VWFpp is constant. The evaluation of the VWFpp/VWF:Ag ratio has been claimed to be useful to differentiate between congenital and acquired forms of the disease [79,81] and also to monitor, in some AVWS patients, remission of the disease [82]. After the exclusion of the possibility of an AVWS, a significantly increased VWFpp/VWF:Ag ratio along with a reduced VWF:Ag level may support the presence of a genetic defect in the VWF responsible for VWD [80] or in other genes responsible for VWF clearance [83,84]. VWD type 1 “Vicenza” is characterized by low levels of VWF:Ag, at about 10 IU/dL, but normal intraplatelet VWF content. In these patients the reduced plasma VWF levels have been attributed to an increased clearance of the “Vicenza” VWF [85], which is markedly higher than the clearance rates of all other VWF variants [79]. Therefore, the evaluation of the VWFpp/VWF:Ag ratio allows to easily detect the “Vicenza” variant [86]. Some authors have proposed to classify as VWD type 1C all the VWF type 1 variants associated to an increased clearance of VWF [80], even though the rate of this increased clearance seems to be too common among the VWF variants to be used to identify a specific subtype [87].

9.6. VWF intraplatelet content The evaluation of intraplatelet VWF requires a fresh blood sample. Platelet are isolated using a density gradient, counted using a blood cell counter and lysed in order to extract VWF stored in the platelet αgranules [92]. The sample so obtained is divided in aliquots and frozen using liquid nitrogen. The intraplatelet VWF is then evaluated for the VWF:Ag and platelet-dependent VWF activity content and their multimeric pattern. Intraplatelet VWF is characterized by ULVWF multimers similarly [93] to those that appear in the circulation in a normal subject after the infusion of desmopressin. On the basis of intraplatelet VWF determination three categories of patients have been reported: platelet normal, those with both normal VWF:Ag and platelet-dependent VWF activity levels; platelet low, those with both reduced VWF:Ag and platelet-dependent VWF activity levels; platelet discordant, those with normal VWF:Ag and reduced platelet-dependent VWF activity levels [94]. Type 2B, type 1 “Vicenza” and type 2N have a normal platelet VWF content. Most type 2A have a low VWF platelet content and VWD type 2M has a discordant VWF platelet content. VWD type 1 has normal or low VWF platelet content. The evaluation of VWF platelet content can also be helpful to differentiate between the congenital and the acquired form of the disease, because in the latter platelet VWF is invariably normal.

9.5. Ristocetin-induced platelet agglutination (RIPA)

9.7. Molecular analysis of the VWF gene

RIPA is evaluated using an aggregometer and requires the use of a fresh blood sample, that is centrifuged shortly after its collection to obtain a plasma rich in platelets (PRP). The PRP is normalized to 250,000 platelets/μL using the patient plasma poor of platelets and then used to evaluate the threshold value of ristocetin able to induce 30% of platelet agglutination. Normal range is between 0.8 and 1.2 mg/ mL of ristocetin [88]. The RIPA is considered enhanced when the

The genetic analysis of the VWF is not necessary to obtain a diagnosis of VWD if the patient has been extensively investigated at the biochemical level. However, if some of the biochemical assays are not available, like the VWF:FVIIIB or the RIPA assays, the molecular approach might be conclusive to reach the diagnosis. Nevertheless, to 6

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distinct VWD types (IIA, IIC, IID and IIE) [66]. All these type 2A forms segregate with a dominant pattern, with the exception of type 2A(IIC) that segregate in a recessive manner. In the most common form of VWD type 2A(IIA) the variants are located in the A2 domain which contains the ADAMTS13 cleaving site [102]. The second most common form of type 2A(IIE) is due to variants in the D3 domain (mainly exon 25–27), important in the VWF multimerization process [67]. A rare form of VWD type 2A, previously reported as IID, is due to variant in the CK domain (encoded by exons 51 and 52) important for the dimerization process [68]. Another rare form of VWD type 2A(IIC) is due variants in the propeptide important for the multimerization process (domains D1 and D2 encoded by exons 2–17) [103]. These patients can be either homozygous for a mutation in the propeptide or compound heterozygous with a mutation in the propeptide and another causing a null allele elsewhere in the VWF.

perform the genetic analysis also in well characterized patients is a useful confirmation of the biochemical diagnosis and the identification of the causing-disease mutation/s is important for genetic counseling and, in the VWD type 3 patients, for prenatal diagnosis. In the past the huge size of the VWF (178 Kb with 52 exons) has hamper genetic analysis in VWD patients, and this had particularly affected the identification of mutations in the type 1 and 3 variants, where the defects are scattered throughout out the whole gene, whereas it did not affect so the type 2 variants, that present with mutations clustered in specific VWF domains [15]. A further complication of the VWF (chromosome 12) sequence analysis is due to the presence of a partial pseudogene on chromosome 22, spanning from exon 23 to exon 34 with 97% homology with the gene [95]. Nevertheless, nowadays technologies like Next generation sequencing (NGS) analysis [96] have become available in many laboratories, allowing investigation of VWF mutations at an affordable price. In order to identify the VWF mutations in both VWD types 1 and 3 patients is necessary to investigate the entire coding region of the gene, including the bordering regions of the introns, the 5′ and 3’ UTR and the most adjacent portion of VWF promoter. VWD type 3 segregates in a recessive manner, so that in these patients two mutations are expected to be found, i.e. the same mutation in both alleles (homozygous) or two different ones (compound heterozygous). VWD type 1 segregates typically in a dominant manner, therefore only one mutation is expected. However, at variance with type 3 where the genetic defects are found in almost all cases, in type 1 the mutations are not identified in about 30% of the patients [97,98], predominantly in those with only modestly reduced VWF levels (VWF:Ag > 30 IU/dL). In addition, most of the mutations identified in type 3 are responsible for null alleles, so that they are easily identified as disease-causing mutations, whereas in type 1 the mutations identified are mainly missense mutations and their association with the disease is not always certain. The use of Multiplex Ligation-dependent Probe Amplifications analysis [99] have been recommended in previously untreated VWD type 3 patients, because the identifications of large gene deletions has been reported to predict the development of VWF inhibitors after replacement therapy [100]. In the case of the VWD type 2 variants it is possible to limit the genetic analysis to the exons encoding for the VWF domains identified using the biochemical approach (Fig. 1). The large majority of the mutations that cause type 2 variants are found in the exon 28 and are mainly missense mutations [101]. Exon 28 is the largest exon (1379 bp) of the VWF and encodes mainly for the A1 and the A2 domains. The type 2B is inherited with a dominat pattern and all the reported variants are located between p.Glu1260 and p.Gly1479 in the A1 domain. If during the RIPA performance the platelet/plasma mixing tests were not performed, the molecular analysis of patients diagnosed with type 2B should always be done in order to establish if the enhanced RIPA was due to a gain-of-function-mutation in the VWF A1 domain (VWD type 2B) or in the GPIbα (Pseudo-VWD) [91] because this distinction is relevant for patients treatment. Also type 2M is inherited with a dominant pattern. The variants that present with a reduced platelet-dependent VWF activity in comparison to VWF:Ag levels are all due to mutations in the A1 domain, whereas most variants with a reduced VWF:CB/VWF:Ag ratio are located in the A3 domain (encoded by exons 29-32), although some of them have been reported also in the A1 domain (collagen binding defects to collagen type IV and VI) [72]. VWD type 2N is due to mutations that cause a reduced capacity of VWF to bind FVIII and are inherited with a recessive pattern. The defects that cause a reduced VWF:FVIIIB are located in the D′D3 domains (encoded by exons 17-25). VWD type 2N can be due to a homozygous mutation in the D′D3 domains or to two mutations (compound heterozygous), one in the D'D3 domains and another responsible for a null allele elsewhere in the VWF. Due to the different pathogenetic mechanisms that can cause VWD type 2A variants the identification of the mutations might be more complex. The VWD type 2A variants are characterized by a variable loss of HMWM, which can be due by mutations in different functional domains and in the previous classification were indicated as

10. Laboratory strategy to the diagnosis of VWD If the evaluation of patient personal and family bleeding history leads to suspect VWD, laboratory tests should be performed to confirm or excluded this hypothesis. From the laboratory point of view the tests employed for diagnosis are performed in four steps (Table 2), requiring that the patient undergos twice blood withdrawal. Hemostasis screening tests are performed at the same time of the first level tests. At this point in time patient plasma samples are aliquoted, frozen at −80 °C and kept for further assays. If further tests are required most of the second level tests can be performed using the patient frozen samples. However, RIPA and the intraplatelet VWF levels require a fresh blood withdrawal. In this occasion the first level tests are repeated to confirm the previous results. Even though we tend to state that first level tests allow to identify the three VWD types (1, 2 and 3), this is not completely accurate. Conclusive results can be reached for normal subjects and type 3 patients, whereas the distinction between type 1 and type 2 requires further testing. Several possible situations can be generated by this set of assays, the more common being reported in Fig. 2(A). In subjects with normal VWF levels and both ratios > 0.6 (Fig. 2, case I), the diagnosis of VWD should be excluded and no second level tests should be performed. However, VWF values at the low level of the normal range should be reevaluated in a second blood sample due to biological variability and thus to avoid to miss the diagnosis of mild forms of VWD. Second level tests should be performed in the remaining cases to reach a final diagnosis. Fig. 2(B) shows the evaluation using the VWF:FVIIIB, multimer analysis, VWF:CB, and RIPA assays in the different cases presented in Fig. 2(A). The use of multimer analysis and RIPA are mandatory in the majority of cases, whereas the VWF:FVIIIB and VWF:CB are more specific to identify the variants with type 2N or type 2M with collagen binding defect, respectively. The molecular analysis of VWF, not necessary in patients fully characterized biochemically, can be performed to further confirm patients diagnosis and to provide a genetic counseling to the patient and his/her family [91]. 11. Conclusion The heterogeneity of VWD requires several assays in order to obtain a complete characterization of VWF plasma levels and functional activity and thus an accurate VWD diagnosis, that is important in order to establish the most adequate treatment for the patient and to ensure an optimal counseling. Although not all the second level tests are needed to obtain patients diagnosis, we try to evaluate patients using the majority of the available assays. To achieve a correct VWD diagnosis it is important to assess the consistency of the results obtained with the different assays, that sometimes need to be repeated more than once. Because molecular biology techniques like next generation sequencing are becoming more commonly available in the diagnostic laboratories, the use of this technique may soon change drastically our approach to 7

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Fig. 2. Panel (A) features the main possible results that can be obtained using VWD first level tests. The full stop “●” reported after the ratios (FVIII:C/VWF:Ag and VWF:RCo/VWF:Ag) indicates that no further evaluation is required. Panel (B) shows the use of VWD second level tests to achieve a VWD diagnosis. Case I: If all three assays give normal values, the ratios between the VWF:RCo/VWF:Ag and the FVIII:C/VWF:Ag should also be considered. If both ratios are > 0.6 the diagnosis of VWD should be excluded, even though VWF values at the low levels of the normal range should be reevaluated in a second blood sample due to biological variability. Case II: Reduced levels of FVIII:C with VWF:Ag and WF:RCo within the normal range, associated with VWF:RCo/VWF:Ag > 0.6 and FVIII:C/VWF:Ag ≤0.6 suggest the diagnosis of hemophilia A or VWD type 2N. Case III: Mildly reduced levels of VWF:Ag and WF:RCo, (< 50% > 30%) with a normal or mildly reduced FVIII:C, associated with VWF:RCo/VWF:Ag > 0.6, suggest a diagnosis of “low VWF”. However, the diagnosis of a mild type 2B or type 2M with reduced collagen binding affinity is also possible. Case IV: Reduced levels of VWF:Ag and WF:RCo (< 30%), with a normal or reduced FVIII:C, associated with VWF:RCo/VWF:Ag > 0.6 suggest a diagnosis of VWD type 1. However, also in this case the diagnosis of a mild type 2B or type 2 M with reduced collagen binding affinity is possible. Case V: Reduced levels of VWF:RCo with a normal or reduced VWF:Ag and a normal or reduced FVIII:C, associated with VWF:RCo/VWF:Ag < 0.6, suggests a diagnosis of type 2 (2A, 2B or 2M). Case VI: undetectable levels of VWF:Ag (< 1%) with a FVIII:C < 10% suggests a diagnosis of type 3. In all the above cases, regardless the values obtained with the VWF:Ag and VWF:RCo assays (reduced, mildly reduced or normal), a VWF:RCo/VWF:Ag ratio < 0.6 suggests a diagnosis of type 2 and further tests should be performed, as reported in case V, to carry out the diagnosis. In addition, to proper fulfill the diagnosis of type 2 M with collagen binding defects or that of type 1 the multimer analysis should also be performed to confirm the presence of a normal or nearly normal VWF multimeric pattern. FVIII:C, factor VIII coagulant activity; VWF:Ag, VWF antigen; VWF:RCo, VWF ristocetin cofactor activity or any other assays that measure the platelet-dependent VWF activity; VWF:FVIIIB, VWF binding to FVIII assay; HMWM, high molecular weight multimers; VWF:CB, VWF collagen binding; RIPA, ristocetin-induced platelet agglutination. Type 2M CB, VWD type 2M with collagen binding defect; Type 2B* pertains to VWD type 2B variants with a full set of HMWM [62,63]. VWD type 2B diagnosis should also be confirmed by molecular analysis in order to exclude platelet-type VWD.

Acknowledgements

the diagnosis of VWD. Nevertheless, a few plasma-based assays will continue to be needed, in order to prove at the biochemical level the effects of the mutation/mutations identified.

The authors would like to thank Prof. P.M. Mannucci for his critical advice and L.F. Ghilardini for the illustration work. We also thanks Prof. U. Budde, who kindly provided the image of multimer analysis of the VWD type 2A (IID).

Author contributions

References

LB wrote the manuscript. FP critically revised the manuscript. Both authors approved the submitted and final version of the manuscript.

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Declaration of Competing Interest LB stated that he had no interests which might be perceived as posing a conflict or bias. FP has received honoraria for participating as a speaker at educational meetings organized by Ablynx, Grifols, Novo Nordisk, Roche, Shire and Sobi. She received consulting fees from Kedrion and she is member of the Ablynx scientific advisory board.

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