von Willebrand Factor Disease (vWD) Profile 

von Willebrand factor (VWF) is a large, complex plasma glycoprotein essential for normal hemostasis^.2,5,11-14 Because VWF attaches platelets to damaged vessel walls after injury, any change in level or function of this protein may lead to a compromise in primary hemostasis.¹⁵ VWF also acts as a carrier protein for coagulation factor VIII (FVIII), protecting FVIII from premature degradation.^15,16 Therefore, a deficiency or defect in VWF may also hamper secondary hemostasis. von Willebrand disease (VWD) is the most common congenital bleeding disorder that can present as a VWF deficiency in type 1 or the more severe type 3 VWD or as a qualitative defect in type 2 VWD. Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those of congenital VWD.^17-20 However, unlike the inherited disease, AVWS occurs in persons with no personal and family history of bleeding and is often associated with a variety of underlying conditions, most frequently lymphoproliferative, myeloproliferative and cardiovascular disorders.

VWD is characterized by a heterogeneous clinical presentation, ranging from minor bleeding episodes to severe bleeding problems that require direct hemostatic control.¹⁶ The more symptomatic VWD patients experience excessive mucocutaneous bleeding, including heavy menstrual bleeding, epistaxis, easy bruising, prolonged bleeding from minor wounds and the oral cavity and gastrointestinal bleeding, as well as bleeding after dental work, childbirth, or surgery, with musculoskeletal bleeding also seen in the most severe cases.⁵

VWF is produced, stored and released into the plasma from the alpha granules of platelets and the Weibel Palade bodies of the vascular endothelium. VWF enters the plasma as a series of oligomers containing a variable number of subunits, ranging from a minimum of two to a maximum of around 40, with the largest (high molecular weight multimers (HMWM) having molecular weights in excess of 20,000 kDa.^21,22 Rapid unfolding of HMWM into ultra-long strings occurs with VWF “docking” on the vascular endothelial cells to permit rope-like adhesion to platelets anchoring platelets to sites of injury.⁷ This process leads to platelet activation, release of granule contents, including more VWF and coagulation proteins (Factor V and fibrinogen), and ultimately, platelet aggregation. VWF also delivers FVIII to the injury site, which serves to expedite secondary hemostasis on the activated platelet surface. This leads to the conversion of fibrinogen into insoluble fibrin, and formation of stable platelet plugs.

Functional VWF works by binding to:

• platelets, primarily to glycoprotein Ib (GPIb) receptor, but additionally to GPIIb/IIIa (also known as integrin αIIbβ3);
• the subendothelial matrix, primarily via the protein collagen;
• coagulation factor VIII (FVIII) and protecting this protein from degradation.

VWD can be characterized or “typed” according to the overall deficit of VWF or a relative deficit of specific forms of VWF (especially the HMWM and, to a lesser extent intermediate molecular weight multimers; IMWM) or based on specific functional defects in VWF.^9,12,13,23 Two defined types of VWD encompass patients with diminished levels of fully functional VWF.^2,5

Type 1 VWD: Characterized by reduced levels of functionally normal VWF. This group includes type 1C, which is characterized by an increase in VWF clearance from circulation. Type 1C is a rare subset of type 1 in which there is a reduction of VWF in circulation because more of it is being cleared, as opposed to synthetic dysfunction.^5,24

Type 3 VWD: Characterized by a (virtual) absence of plasma VWF. This is the rarest and most severe type of VWD. It is typically inherited in an autosomal recessive or compound heterozygous pattern.²⁵ Patients with type 3 VWD are prone to severe bleeding that often mimics bleeding in hemophilia. Due to their lack of VWF, these patients have low factor VIII levels because factor VIII is not stabilized in plasma by VWF.²⁶

Another group of patients present with “dysfunctional” VWF, with or without reduction in plasma VWF antigen levels. All type 2 subtypes except for 2N have an autosomal dominant inheritance pattern.11 Type 2 VWD is generally subdivided into four distinct disorders:

Type 2A VWD: Characterized by a reduction of high molecular-weight multimers (HMWM) of VWF due to a genetic defect compromising the formation of HMWM from VWF monomers.

Type 2B VWD: Characterized by hyper-adhesive VWF where VWF “spontaneously” binds to platelet GPIb (in the absence of injury), leading to clearance of both HMWM and platelets from circulation. These individuals have lower residual VWF activity with a multimer pattern similar to type 2A accompanied by mild thrombocytopenia.²⁷ Type 2B VWD individuals may have a mild thrombocytopenia, or a normal platelet count with either normal-sized platelets²⁸ or giant platelets.²⁹ Type 2B VWD can be differentiated from other forms of type 2 VWF by an elevated response to ristocetin in a ristocetin-induced platelet aggregation (RIPA) assay. Recent guidelines suggest genetic testing should be undertaken to identify this genotype.⁵

Type 2M VWD: This VWD “type” refers to a heterogeneous group with defective VWF activity not associated loss of HMWM.^14,30 Most cases of type 2M VWD express mutations that lead to loss of GP1b binding, but often these mutations have limited or no effect on collagen binding.³¹ However, cases with defective collagen binding or defects in both GP1B and collagen binding occur as well.

Type 2N VWD: Characterized by a dysfunction in VWF FVIII activity binding, which then leads to increased degradation of FVIII and loss of plasma FVIII activity and leads to disease state phenotypically similar to hemophilia A.

Types 2A, 2B and 2M have an autosomal dominant inheritance while type 2N penetrates by an autosomal recessive trait.^14,32

Pseudo-VWD: Another congenital condition that should be considered in the differential diagnosis of VWD is “platelet type (PT)” or pseudo-VWD.^33,34 Although not really VWD, since defects lie in the platelet GPIb receptor, consideration of PT-VWD testing is required, since patient presentation is phenotypically similar to type 2B VWD. This exceedingly rare gain-of-function defect of GPIb leads to “spontaneous” binding to normal plasma VWF, leading to clearance of both functional HMWM and platelets from circulation. These patients have diminished VWF activity and a macrothrombocytopenia of varying degrees.³⁵

Acquired VWD: Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those of inherited von Willebrand disease.^17,36 However, unlike the inherited disease, AVWS occurs in persons with no personal and family history of bleeding and is often associated with an underlying disease. Most cases are due to an increased plasma clearance of VWF caused by such mechanisms as antibodies, cell adsorption or increased proteolysis.^1,37-39 AVWS is associated with lymphoproliferative/ hematological malignancy, myeloproliferative, other neoplastic and autoimmune disorders along with or various other causes (plasma-mediated hyperfibrinolysis, glycogen storage disease, uremia, hypothyroidism).^40-43 AVWS is associated with cardiovascular disorders, including aortic stenosis (Heyde syndrome), congenital heart defects, hypertrophic obstructive cardiomyopathy, tetralogy of Fallot, pulmonary hypertension, mitral regurgitation, and the use of left ventricular assist devices.^18,44-51 AVWS in cardiovascular conditions has been linked to intravascular shear-induced loss of the highest-molecular-weight multimers VWF.⁵²

The characterization of patients with VWD into one of these “subtypes” has clinical relevance since different therapeutic/management options may apply according to both type and severity of VWD.^53,54 In brief, type 1 VWD, especially if only mild (i.e., only a slight loss of VWF), can be treated, at least for a short period, with a nontransfusional form of therapy using desmopressin (DDAVP), which facilitates release of stored reserves of VWF. However, most forms of type 2 VWD will require VWF replacement therapy, since DDAVP will only act to release “abnormal” VWF.

VWD diagnosis relies on a clinical bleeding assessment, a preliminary laboratory evaluation, a quantitative assessment of VWD levels, a qualitative assessment of VWF function, and, if applicable, specialized tests to determine the subtype of VWD.55 Laboratory screening for VWD requires the performance of three assays.^2,5,55 These are a VWF “antigen” (VWF:Ag) assay, a VWF GPIb binding activity (VWF:Ac) assay and the FVIII activity (FVIII) assay. This format of 3-test panels is recommended by the latest American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia 2021 guidelines.⁵ Additional assays (not included in this profile) are required to specifically classify patients with type 2 VWD.

Von Willebrand Factor Antigen (VWF:Ag): The VWF:Ag assay is a primary assay that identifies the level of VWF but detects both active and inactive (or nonfunctional) VWF.⁵⁶

Von Willebrand Factor Activity (VWF:Ac): The VWF:Ac assay measures platelet GPIb binding activity. This assay is sensitive to the presence or absence of HMWM and IMWM forms of VWF, which accordingly express high or intermediate VWF:Ac levels. The Labcorp VWF:Ac assay employs a monoclonal antibody that targets the region of the VWF molecule that binds to the glycoprotein 1b receptor (GP1B binding) as a measure of VWF activity.⁵⁷

The assay employs a recombinant GP1b fragment genetically modified to include two gain-of-function mutations (GP1bM). These gain-of-function mutations allow for the measurement of VWF binding to GP1b without addition of ristocetin.^55,58

VWF:Ac/VWF:Ag Ratio: The VWF:Ac/VWF:Ag ratio will be normal (>0.7) when the VWF in the patient’s plasma is fully functional with regard to GP1B binding activity but will be reduced due to a compromising genetic defect in the GP1B binding locus of the VWF and/or due to a reduction in HMWM as a fraction of total VWF.5,59 VWF:Ac/VWF:Ag is >0.7 in normal individuals and in patients with type 1 VWD, where VWF may be low but the VWF:Ac activity of the existing VWF is preserved. Ratios are generally not generally useful in type 3 VWD due to the imprecision of the component measurements at low VWF levels. VWF:Ac/VWF:Ag will be low (<0.7) in patients with type 2A or 2B or PT-VWD, since the HMWM fraction of VWF is reduced. VWF:Ac/VWF:Ag is also reduced in the majority of type 2M VWD cases because the HMWM present is functionally compromised. Type 2N disease would be expected to have a VWF:Ac/VWF:Ag ratio >0.7.

It must be noted that in some individuals, reduced ratios can be calculated despite normal VWF activity and VWF:Ag levels; this has been reported in type 2B VWD60 and AVWS.⁶¹

Factor VIII (FVIII): Testing for FVIII level is typically measured in patients with a substantial bleeding history that is suspicious for hemophilia. Given that VWF protects and stabilizes FVIII, testing for FVIII activity is also required for a complete VWD workup.²³ Low levels of FVIII combined with low levels of VWF compounds bleeding risk (causing defects in both primary and secondary hemostasis). Notably, FVIII is significantly lowered (5% to 15%) in type 2N VWD where a dysfunction in VWF FVIII binding leads to increased degradation of FVIII and loss of plasma FVIII activity.^11,62 Very low FVIII levels VWD (1% to 10%) are found in patients with type 3 VWD.^11,62 A normal level of FVIII does not always exclude VWD (FVIII levels will be normal in many patients with “mild” type 1 VWD and also in some patients with type 2B and 2M VWD), and an abnormal FVIII does not always establish a diagnosis of VWD (hemophilia A is actually more likely).

FVIII/VWF:Ag Ratio: Incorporation of test ratios provides context around specific VWF activity relative to total VWF concentration.²³ In healthy individuals and in those with types 1, 2A, 2B, 2M and 3 VWD, levels of FVIII:C are relatively concordant (often a little higher) with VWF:Ag. This is reflected by a FVIII/VWF:Ag ratio >0.7. However, individuals with type 2N VWF or hemophilia A should have a FVIII/VWF:Ag ratio <0.7. It should be noted that ratios in patients with calculated ratios in type 3 VWD are not clinically valuable as the denominator will be very close to zero. It is important to consider that a low ratio may potential represent a preanalytical issue due to the highly labile nature of the factor VIII molecule (see limitations).

Results Interpretation

Scenario 1: All results (VWF:Ag, VWF:Ac and FVIII) normal—VWD/AVWS can largely be excluded (>90% confidence level). This is especially true if VWF levels are >100 U/dL.^63,64,70 Two caveats prevent 100% confidence for exclusion:

• There are some rare cases of VWD, notably 2M VWD, that can only be identified by performing VWF:CB assays, these cases will be missed.^65,66
• VWF is an acute phase protein, which increases following exercise, anxiety, infection, and during pregnancy. Thus, normal VWF levels occasionally mask (mild) type 1 VWD. The strategy, in patients with relevant clinical history, is repeat testing performed on fresh samples following exclusion of these issues (i.e., no exercise, anxiety, infection, pregnancy). Anxiety/stress related increases in VWF are particularly relevant in pediatric patients.

Scenario 2: Detectable VWF:Ac results between 5% and 50%; with a concordant VWF:Ac/VWF:Ag Ratio (>0.7)— Patients with type 1 VWD present with reduced levels of functionally normal VWF.

• Having low levels of VWF:Ac per se (i.e., below normal reference range cutoff) does not in itself define VWD. The ASH ISTH NHF WFH 2021 guideline recommends that a detectable VWF:Ac level (between 5% and 29%) regardless of bleeding, confirm the diagnosis of type 1 VWD.⁵ Clinical evidence of bleeding/bruising must also be present for patients with a minimally diminished VWF:Ac level (30% to 50%) for the diagnosis of type 1 VWD to be assigned.⁵ A VWF:Ac <30 IU/dL is usually associated with bleeding symptoms and is likely to be associated with a variant in the VWF gene, but these associations are less strong for reduced VWF activities between 30 and 50 IU/dL.⁶⁷ VWF activity between 30 and 50 IU/dL in isolation may be insufficient to result in significant bleeding, although some individuals with VWF activity between 30 and 50 IU/dL do have significant bleeding symptoms: this is likely to reflect the interaction with additional abnormalities in the hemostatic pathway, including mild platelet defects.⁶⁷
• Individuals with VWF levels between 30 and 50 U/dL, with concordant VWF activity/Ag ratio but without appropriate clinical history, can be assigned to a group of “low VWF as a risk factor for bleeding,” but not to VWD status.⁵
• A decrease in VWF survival (or increased VWF clearance) is thought to account for 15 to 20% of type 1 VWD cases.⁵ The recent guideline suggests using a desmopressin trial with 1- and 4-hour post-infusion blood work to confirm increased VWF clearance for patients with VWD suspected of type 1C.⁵ The response to desmopressin is considered to reflect increased VWF clearance if a four-hour post-infusion time point shows a >30% decrease from the peak VWF level (see Labcorp Test No. 184040).^5,24 The accurate identification of patients who have type 1C VWD has management implications, as these patients may require VWF concentrate to treat/prevent bleeds.⁵

Scenario 3: Detectable VWF:Ac results between 5% and 50%; with a discordant VWF:Ac/VWF:Ag Ratio (<0.7)—This pattern can occur in any of the following: Type 2A, 2B, 2M or PT-VWD. Additional testing is required for the classification of VWD type in these patients.^2,5

• To differentiate between type 2A/2B and 2M, current diagnostic guidelines recommend either VWF multimer (VWF:MM) analysis or the VWF collagen binding activity (VWF:CB) with ratio to VWF antigen (VWF:CB/VWF:Ag ratio).^5,68 This differentiation carries clinical significance as it has been proven that patients with diminished VWF:Ac and VWF:CB tend to have a more severe bleeding phenotype.⁵
• Acquired VWD is often associated with a decrease in VWF:Ac/VWF:Ag ratio and diminished high molecular weight multimers.^{16,17,36,52,69}
• Ristocetin induced platelet agglutination (RIPA) can be performed to identify or exclude 2B or PT VWD (both show response to low ristocetin concentrations).^5,70,71 However, the recent guideline suggests targeted genetic testing over RIPA to diagnose type 2B VWD for patients suspected of type 2A or 2B in need of additional testing.⁵ Genotyping may be helpful in confirming VWD subtypes (including type 2B, 2M, and 2N disease) or PT-VWD when results might affect therapeutic decisions.^5,68,72,73
• For type 2M VWD, the most common forms have low VWF:Ac/VWF:Ag but normal VWF:CB/VWF:Ag.
• Some publications recommend genetic analysis of VWF for all cases yielding consistently low VWF:Ac/VWF:Ag and/or VWF:CB/VWF:Ag ratios unless the defect is related to AVWS.¹² Notably, AVWS can be considered as a diagnosis when there is new onset of bleeding with no preceding history of bleeding with challenge, in the setting of cardiac valve disease, monoclonal gammopathy or myelo/lymphoproliferative disorders.¹² Consider molecular analysis (Labcorp’s von Willebrand Disease Genetic Analysis [630468]) to confirm VWD type.

Scenario 4: Low FVIII activity with a low FVIII /VWF:Ag ratio (<0.7)—This pattern can be seen in patients with Hemophilia A and type 2N VWD.

• Type 2N VWD can be mistaken for hemophilia A because of the low FVIII level, with critical treatment implications: patients with hemophilia A are treated with FVIII concentrate; however, type 2N VWD patients require VWF replacement therapy to prevent/treat serious bleeds.⁵ In contrast to all other type 2 and type 1 VWD, type 2N is autosomal recessive and accurate diagnosis is needed to inform genetic counseling within families.⁵
• However, it should be noted that factor FVIII is particularly susceptible to preanalytic degradation. Repeat testing on fresh samples for confirmation, ensuring appropriate collection, storage, and transportation of samples is recommended to confirm initial low results by repeat testing on a fresh sample. If test findings are confirmed, hemophilia A and 2N VWD can be differentiated using either a VWF:FVIIIB assay or by genetic analysis of F8 and VWF genes, respectively.^5,77 Consider molecular analysis (Labcorp’s von Willebrand Genetic Analysis [630468] and Factor VIII (Hemophilia A) Genetic Analysis [630619]) to confirm genotype.

Scenario 5: VWF:Ag result very low (<5 U/dL)—The patient may have type 3 VWD.

• Repeat testing on fresh samples for confirmation, ensuring that tests accurately detect VWF levels <5 U/dL before identifying patients as type 3 VWD.² Consider molecular analysis VWD type (von Willebrand Disease Genetic Analysis [630468]) to confirm VWD type.