von Willebrand disease (VWD) is the most common bleeding disorder, with a prevalence of up to 1%.^1,2 VWD is caused by either a quantitative or qualitative defect of von Willebrand factor (VWF), which mediates platelet adhesion by binding to the GP1b platelet glycoprotein and to collagen on injured blood vessels. Circulating VWF also binds to factor VIII, protecting it from proteolysis.³ VWF, synthesized by endothelial cells and megakaryocytes, circulates as multimeric glycoproteins ranging in size from 500,000 to >20 million Daltons.^3,4 High-molecular-weight (HMW) VWF multimers bind GP1b and collagen more strongly, thus mediating platelet adhesion more effectively than lower molecular weight forms.⁵ In VWD, reduced VWF levels or activity causes inadequate platelet binding and decreased protection of factor VIII from proteolysis, resulting in impaired hemostasis.

Three forms of VWD have been described: inherited (the predominant form), acquired (also known as acquired von Willebrand syndrome), and platelet-type. Inherited VWD is further divided into 3 types (types 1, 2, and 3) based on pathophysiology (Table 1).^3,6,7,13

Type 1 VWD, accounting for approximately 70% of inherited VWD cases, is characterized by a partial deficiency of VWF (10% to 40% of normal) due to decreased production or release.² A subset of type 1 VWD caused by increased VWF clearance has also been identified.⁸ Individuals with type 1 VWD may be asymptomatic or have mild symptoms (eg, bleeding from gums or heavy menstrual cycles) until a severe injury or surgical procedure precipitates a significant bleeding episode.^2,3

Type 2 VWD, which accounts for approximately 20% of inherited VWD cases, is characterized by defective VWF function with normal or marginally decreased circulating VWF levels.² Affected individuals tend to have more severe bleeding symptoms than those with type 1 VWD.^2,3 Several type 2 VWD subtypes have been identified, characterized by distinct abnormalities of VWF processing, multimeric composition, and platelet binding (Table 1). Type 2A VWD involves a deficiency of high and intermediate molecular weight (HMW) VWF multimers because of defects in multimerization or increased susceptibility of HMW multimers to breakdown by the ADAMTS 13 protease.³ Individuals with type 2B VWD have HMW VWF multimers that bind platelets with abnormally high affinity, causing a deficiency of circulating HMW multimers in plasma. In type 2M VWD, VWF exhibits normal multimeric composition but defective platelet binding. Type 2N VWD is a variant characterized by defective binding of factor VIII to VWF, which causes decreased factor VIII activity similar to that observed in hemophilia.³

Type 3 VWD, accounting for approximately 10% of inherited VWD cases, is characterized by an almost complete deficiency of VWF and very low levels of factor VIII.² Affected individuals have severe bleeding that can be life-threatening if not recognized and treated.^2,3

Acquired VWD is associated with lymphoproliferative or myeloproliferative disorders, malignancies, immunologic diseases, circulatory dysfunction such as aortic stenosis, and other medical conditions (eg, hypothyroidism); it is also sometimes drug-associated.^9,10 In acquired VWD, VWF is produced normally but is rapidly removed from circulation by tumor cell adhesion, VWF antibody-mediated disruption of large multimers, or proteolytic digestion. Up to 20% of individuals diagnosed with VWD may have an acquired form of the disorder.^9,10

Platelet-type VWD is uncommon and characterized by thrombocytopenia, the loss of large multimers, and a gain of function of the von Willebrand binding protein located on the surface of platelets.¹¹

Diagnosing VWD is important for identifying individuals who are at risk of excessive bleeding.^12,13 Diagnosis usually begins with a clinical assessment that includes taking a personal history of bleeding symptoms and a family history of bleeding disorders. Typical VWD symptoms are abnormal bleeding from the skin and mucous membranes, including prolonged or unusually severe epistaxis; easy bruisability; gingival bleeding; prolonged bleeding from minor wounds or dental extraction; and prolonged and heavy menstrual bleeding (Table 2).¹³ The severity of bleeding, however, does not always correlate with the degree of VWF deficiency, and symptoms may vary among patients with the same type of VWD as well as among family members.^2,6,14 Many affected individuals with inherited VWD will in fact have a negative family history due to incomplete penetrance. Additionally, it is important to rule out the use of drugs that alter clotting function in individuals suspected of having VWD (eg, anticoagulants, antiplatelet drugs, non-steroidal anti-inflammatory drugs [NSAIDs], and diuretics).^13,15

Once VWD has been diagnosed, disease classification is crucial because treatment and bleeding risk vary with type. Treatment for type 1 VWD is desmopressin (DDAVP^®), which induces the release of stored VWF from endothelial cells. DDAVP may also be effective in treating some individuals with types 2A and 2M VWD, but it is ineffective in type 2N and type 3 disease. DDAVP is also not indicated for type 2B and platelet VWD. VWF/factor VIII complexes are used to treat type 2N and type 3 VWD, as well as type 2A or type 2M VWD that does not respond to DDAVP (Table 1). Some individuals with acquired VWD respond to DDAVP; those that do not are typically treated with VWF/factor VIII complexes.^9,10

• Individuals with a history of unexplained menorrhagia, abnormal uterine bleeding, lifelong bruising, unexplained epistaxis, or significant bleeding from minor surgical procedures

• Individuals with a family history of VWD

• Some individuals with autoimmune disorders and/or lymphoma/myeloma and new-onset bleeding

Tests available to assist in diagnosis of von Willebrand disease are listed in the Appendix.

Testing typically begins with a complete blood count (to assess hemoglobin, hematocrit, platelet count, and morphology) and measurement of prothrombin time (PT) and activated partial thromboplastin time (aPTT). Optional thrombin time or fibrinogen tests may also be performed initially.¹³ Individuals with VWD will usually have normal PT, platelet count, thrombin time, and fibrinogen level. Some individuals may have prolonged aPTT that corrects in a mixing study. While a prolonged aPTT is consistent with VWD, the aPTT may be normal in untreated mild or moderate disease and alone cannot exclude VWD.^2,16 It will be prolonged in type 2N and type 3 disease because of very low levels of factor VIII caused by a deficiency in VWF levels or activity, and may be decreased in platelet-type VWD. Consequently, individuals with a prolonged aPTT and individuals with a strong personal and/or family history suggestive of VWD should be tested for VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and factor VIII activity (Figure 1).

A decreased level of VWF:Ag, VWF:RCo, and/or factor VIII clotting activity is consistent with VWD and warrants additional tests to identify the type of VWD; these tests may include multimeric analysis, the VWF collagen binding assay (VWF:CB), and the VWF:factor VIII binding activity assay (VWF:FVIIIB; von Willebrand Disease Type 2N Panel).¹⁶ VWF:CB assay results are reported as the ratio of collagen-bound VWF to VWF antigen. A ratio <0.5 is consistent with types 2A and 2B VWD, whereas a normal ratio (≥0.5) is associated with types 2M and 2N VWD.¹⁷ The factor VIII:VWF binding activity ratio is typically <0.73 in individuals with type 2N. Table 1 summarizes the expected results of tests used in the diagnosis and typing of inherited VWD. There is no definitive pattern of test results in individuals with acquired VWD.

von Willebrand disease mutation analysis may be useful to differentiate hemophilia A from type 2N VWD, distinguish the various type 2 subtypes, and counsel individuals with type 3 VWD.⁶

Quest Diagnostics offers 2 panels (von Willebrand Comprehensive Panel and von Willebrand Comprehensive Panel 2) that include key tests for VWD diagnosis and typing, along with an interpretation of results and additional comments (Figure 2). In both panels, aPTT is used to determine clotting time and VWD is diagnosed by assaying VWF:Ag, VWF:RCo, and factor VIII clotting activity. To help differentiate VWD type 2 subtypes, both panels include an analysis of plasma VWF multimeric composition; in addition, Panel 2 includes a VWF:CB assay.

Activated Partial Thromboplastin Time (aPTT)

The aPTT will be prolonged if there is deficiency or an inhibitor of factors in the intrinsic or common pathways, including high molecular weight kininogen (HMWK); prekallikrein; factors V, VIII, IX, X, XI, and XII; prothrombin; and fibrinogen. A prolonged aPTT is also seen in individuals with lupus anticoagulant. In VWD diagnosis, the aPTT is prolonged only if factor VIII is sufficiently decreased.¹³ Most individuals with type 3 or type 2N VWD and approximately 25% to 50% of individuals with type 1 VWD will have a prolonged aPTT. Therefore laboratory tests for VWD should be performed if clinical suspicion is strong, even if aPTT is normal.¹³

Complete Blood Count (CBC)

Hemoglobin and hematocrit may range from normal to markedly decreased, depending on the type and severity of VWD. See also Platelet Count.

DDAVP Response

DDAVP (desamino-8-arginine vasopressin), a synthetic analogue of antidiuretic hormone, is considered the primary treatment for bleeding in individuals with mild VWD (type 1, sometimes types 2A and 2M). DDAVP works by causing the release of VWF from endothelial storage sites. An individual’s therapeutic response to DDAVP is usually tested before prescribing the medication. A positive response is defined as a 2- to 5-fold increase over baseline in VWF antigen, factor VIII, and/or ristocetin cofactor 30 to 90 minutes after the administration of DDAVP. DDAVP is contraindicated in individuals with type 2B VWD due to the possibility of platelet clumping and subsequent increased risk of thrombosis. It is ineffective in individuals with type 2N, type 3, and platelet VWD.

Factor VIII

Factor VIII is an acute phase reactant that is produced at higher levels during periods of stress, postoperatively, and in inflammatory conditions; elevated levels are also found at birth and during pregnancy. Increased factor VIII levels are associated with increased risk for venous and arterial thrombosis. Because binding by VWF normally protects factor VIII from proteolysis, decreased VWF levels lead to decreased levels of factor VIII. Mild VWD is typically associated with factor VIII levels in the low normal range; type 2N VWD is associated with low factor VIII levels (1% to 40% of normal);^18,19 and type 3 VWD is associated with very low levels (<10%).³ Decreased levels are also associated with hemophilia A and specific factor VIII inhibitors (antibodies).

Mixing Study

When the aPTT is prolonged, a mixing study is often performed to determine if abnormal clotting is caused by an intrinsic defect in clotting factors or by an inhibitor. In the mixing study, patient plasma is mixed with normal plasma and the aPTT assay is repeated. A prolonged aPTT that normalizes immediately after mixing with normal plasma, and does not reverse after incubation, indicates an intrinsic or common pathway factor deficiency and is also consistent with VWD. A prolonged aPTT that remains prolonged after mixing with normal plasma, or an aPTT that normalizes immediately after mixing with normal plasma but prolongs after incubation, is usually not associated with VWD. Such aPTT results may indicate the presence of an inhibitor (eg, lupus anticoagulant or a specific clotting factor inhibitor) or excessive fibrinolysis and fibrinogenolysis.

Platelet Count

The platelet count is normal in most individuals with VWD but is decreased in those with type 2B and platelet-type VWD. A normal platelet count in the presence of increased bleeding is consistent with VWD, vasculopathies, connective tissue diseases, and qualitative platelet abnormalities, and warrants further testing for VWD. A normal platelet count and normal bleeding is consistent with fibrinolytic disorders and may be observed in some individuals with VWD; thus, further VWD testing is warranted when clinical suspicion is high.

Prothrombin Time (PT)

The PT measures the time for clot formation after the addition of tissue factor (thromboplastin) and calcium to citrated blood. PT is prolonged with deficiencies of factors II, V, VII, X, and fibrinogen; liver disease; warfarin use; and vitamin K deficiency. PT is within the reference range in individuals with VWD.

Ristocetin Cofactor Activity (VWF:RCo)

Ristocetin is an antibiotic that causes VWF (ristocetin cofactor) to bind to platelets. The ristocetin cofactor assay is an important measure of VWF activity. In this test, patient platelet-poor plasma is mixed with ristocetin and stabilized normal platelets, and platelet agglutination is then measured. Plasma from individuals with normal ristocetin cofactor activity exhibits rapid platelet agglutination. Ristocetin cofactor activity is reduced (<42% of normal) in individuals with most types of VWD, although some individuals with type 2B VWD may have normal activity. Ristocetin cofactor activity may be increased in response to stress and physical exercise and may vary with the menstrual cycle phase.²⁴

von Willebrand Antigen, Multimeric Analysis

The size distribution of VWF multimers, determined by gel electrophoresis, helps establish the type of VWD present. Types 1, 2M, and 2N VWD typically exhibit normal multimer patterns; type 2B and platelet VWD lack high molecular weight multimers; type 2A VWD lacks high and intermediate weight multimers; and type 3 VWD is characterized by absence of all multimers. Acquired VWD is associated with variable multimeric patterns depending on the underlying etiology.

von Willebrand Disease Mutation Analysis

This assay identifies mutations in the VWF gene associated with types 2A, 2B, 2M, 2N, and some forms of types 1 and 3 VWD. This assay is useful for differentiating mild hemophilia A (no VWF mutations, X-linked inheritance) from type 2N VWD (VWF mutations, autosomal recessive inheritance); type 2B from type 2M VWD; type 2B from platelet-type VWD; and type 2A from type 2B VWD (useful due to the relative contraindication of DDAVP in type 2B VWD).^20,21 Mutation analysis can also determine the causative mutation in families with type 3 VWD to help in the management of future pregnancies.⁶ This analysis is less useful in diagnosing type 1 VWD, which has a complex and variable genetic basis.²¹ Frequent mutations associated with each VWD type are described in the ISTH-SSC VWF Online Database (see Related References section). Additional information regarding the use and interpretation of this test may be obtained by calling Quest Diagnostics genetic counselors at 1-866-GENE-INFO (1-866-436-3463).

von Willebrand Disease Type 2N (VWF:Factor VIII Binding Activity; VWF:FVIIIB)

This test, which measures the binding of exogenous factor VIII to a patient’s VWF antigen, is used to identify type 2N VWD. A factor VIII:VWF binding activity ratio of 0.73 to 1.42 is considered normal. A normal ratio may be seen in VWD other than type 2N because factor VIII activity and VWF antigen may be reduced proportionally. In type 2N VWD, factor VIII activity is reduced but VWF antigen levels are normal; the ratio is thus decreased. A ratio <0.73 is consistent with type 2N VWD.

von Willebrand Factor Activity (GP1b-specific EIA; functional VWF)

This assay directly measures the functional activity of VWF and is used to confirm low (<20%) ristocetin cofactor levels. Levels <35% are consistent with VWD, while levels above the reference range (35% to 134%) have no known clinical significance.

von Willebrand Factor Antigen (VWF:Ag)

This test measures the total amount of VWF protein. Levels are decreased in types 1 and 3 VWD. VWF antigen levels may be normal or decreased in types 2A, 2B, and 2M. Levels are normal in type 2N VWD.

Levels of VWF vary by blood type and ethnicity. The mean level of VWF in individuals with blood type O is approximately 30% lower than in individuals with blood types A, B, or AB.²² Higher mean levels of VWF are found in African Americans than in other ethnic groups.²³ Debate exists about whether reference ranges should be specific for blood group type or ethnicity; however, bleeding tendency is primarily related to VWF antigen level and multimeric composition.^2,16

Increased levels of VWF may be seen secondary to stress, inflammation, acute infection, physical exercise, following surgery and estrogen therapy, and during the second and third trimesters of pregnancy.^24,25 VWF levels may also vary with the menstrual cycle phase.²⁴ Serial testing may thus be necessary to confirm or rule out VWD.  

von Willebrand Factor Collagen Binding Assay (VWF:CB)

The activity of VWF is determined by measuring the ability of VWF to bind collagen. The ability of VWF to bind collagen is a function of large multimers; thus, collagen binding activity is abnormal in types 2A and 2B VWD and some acquired VWD cases, but may be normal in types 2M and 2N. Collagen binding is compared to the VWF antigen value by calculating a collagen binding ratio (collagen-bound VWF:VWF antigen). Typically, a ratio ≥0.5 is considered normal, whereas a ratio <0.5 is consistent with VWD.