-Contributed by Abi Kasberg, PhD
Von Willebrand disease (VWD) is an inherited bleeding disease that clinically presents with extended bleeding times following wounds, surgeries, nosebleeds, or during menstruation. VWD affects approximately 0.1% of people and is considered to be the most common bleeding disorder (Keesler and Flood 2017). Von Willebrand factor (vWF) is a multimer protein that functions to bind platelets, bind collagen, and bind FVIII to promote hemostasis and stop bleeding (Keesler and Flood 2017). VWD is caused by deficiencies in vWF. VWD Type 1 or Type 3 captures quantitative deficiencies in vWF such as decreased amounts of vWF protein in the blood. VWD Type 2 consists of qualitative vWF defects and occurs when vWF activity is impaired. Genetic variations of vWF can interfere with platelet binding, collagen binding, and FVIII stability, leading to VWD subtypes and excessive bleeding.
While researching VWD, it is important to consider the impact that VWD has on patient experiences and lifestyles. VWD patients encounter acute bleeding events that can be scary, difficult to manage, and life-altering. Injuries and traumas that may be minor to most individuals can quickly become complicated and life-threatening for individuals with VWD. For example, an ear piercing as a young adult (Kruse-Jarres and Johnsen 2018), vaccination injections as a newborn (Dash 2019), or a toddler’s injury to the lower lip (Echahdi et al. 2017) can trigger severe bleeding and in some cases hemorrhagic shock. In addition to acute episodes of severe bleeding, lengthy hospitalization stays may be required for factor replacement therapy and bleeding control. Health-related quality of life (HRQoL) may be reduced due to limited physical and social capabilities, frequent hospitalizations, and restricted mobility. A research study presented at the National Hemophilia Foundation-sponsored Bleeding Disorders Conference indicated that hospitalized VWD patients that are prescribed factor replacement therapy have an average length of stay of 4 days, with a range of 1-70 days (Khachatryan and Ito 2015).
In short, the pathogenesis, management, and treatment of VWD is complex and comes at a HRQoL cost to patients. There is a clinical need for rapid and reliable data that can be leveraged during bleeding events to quickly identify the cause of bleeding as well as guide treatment options and assess hemostatic function following drug treatment.
Available assays for VWD testing
In the clinic, it is essential to identify the type of VWD due to subtype-specific treatment options. There is a broad range of variability in vWF levels, vWF functionality, interacting components, and symptoms. This creates a complex environment in which to diagnose and treat VWD. Complicated clinical guidelines and algorithms for diagnosis have been recommended by a multidisciplinary panel established by the American Society of Hematology (ASH), the International Society on Thrombosis and Haemostasis (ISTH), the National Hemophilia Foundation (NHF), and the World Federation of Hemophilia (WFH) (James et al. 2021). In 2021, this panel approved eleven new recommendations for the assessment of patients suspected of VWD (James et al. 2021). Provided here is a brief review of some of these recommendations that pertain to the use of tools and assays for VWD diagnosis:
Validated bleeding-assessment tools (BATs) are questionnaires or scoring systems that quantify reported bleeding symptoms in standardized formats. Commonly used BATs include ISTH-BAT, MCMDM-1 VWD, and others. The 2021 panel recommends the use of BATs as an initial screening test only for patients with a low probability of VWD (James et al. 2021). Patients with intermediate and high probabilities of VWD should not rely on BATs to determine blood testing needs and instead use BATs to accompany specific blood testing for use in diagnosis (James et al. 2021).
Pros: The primary benefit of BATs is to identify patients who have bleeding symptoms and therefore may have VWD. Additional advantages include that BATs are standardized and may eliminate unnecessary blood testing for some individuals.
Cons: The risk of relying on BATs for diagnosis and leveraging BATs as gatekeepers for additional testing is that affected individuals may be missed due to lack of reported bleeding symptoms. Individuals most likely to be overlooked through screening with BATs are men and children.
vWF platelet-binding activity assays
Due to the importance of vWF-platelet binding during primary hemostasis, it comes as no surprise that vWF platelet-binding activity assays are a critical step during VWD diagnosis. Ristocetin is an antibiotic that facilitates platelet agglutination in the presence of vWF (Ng et al. 2015). The vWF ristocetin cofactor assay (VWF:RCo) is the historical standard assay used to determine the functional activity of vWF in plasma samples. VWF:RCo has limitations including inadequate inter-laboratory reproducibility and variability in result interpretations. This has led to the VWF:RCo assay being gradually replaced with more accurate replacements. Suggested alternative methods of determining platelet-binding activity of vWF are VWF:GP1bR and VWF:GP1bM instead of VWF:RCo (James et al. 2021). VWF:GP1bR uses recombinant GP1b to trigger vWF binding activity in the presence of ristocetin. VWF:GP1bR is dependent on ristocetin concentrations, and therefore is constrained by the same limitations as VWF:RCo, although VWR:GP1bR does show improvement to data variation (Keesler and Flood 2017). To more accurately measure the ability of vWF to induce platelet binding, the VWF:GP1bM assay has been developed. In this assay, the GP1b domain contains gain-of-function mutations that enable spontaneous binding of vWF to GP1b. VWF:GP1bM measures the ability of vWF to bind platelets, most notably in a manner that is not dependent on ristocetin-induced platelet agglutination (Keesler and Flood 2017).
Pros: VWF:GP1bR and VWF:GP1bM assays produce more accurate and consistent data compared to VWF:RCo. VWF:GP1bR measures the ability of vWF to bind platelets independent from ristocetin concentrations.
Cons: VWF:RCo has limitations due to inconsistent reproducibility and variability in result interpretations. VWF:GP1bR is dependent on ristocetin concentrations.
Collagen binding assays
Collagen binding assays measure the functional ability of vWF to bind collagen (VWF:CB). Collagens I and III bind the A3 domain of vWF, and can be detected using most VWF:CB assays. Collagens type IV and VI bind vWF through the A1 domain. The ratio of VWF:CB to vWF antigen levels (VWF:Ag) can distinguish between VWD Type 1 and Type 2. The 2021 panel suggests that either VWF multimer analysis or VWF:CB/VWF:Ag ratio be used to diagnose VWD Types 2A, 2B, or 2M (James et al. 2021).
Pros: VWF:CB/VWF:Ag ratios of 1 indicate that the individual does not have defective collagen binding abilities. Decreases in the VWF:CB/VWF:Ag ratio of <0.6 indicate collagen binding defects (Type 2M) and/or vWF multimer defects (Types 2A and 2B) (Keesler and Flood 2017). The VWF:CB/VWF:Ag ratio may be used as a surrogate for high molecular weight multimer (HMWM) analysis, particularly when diagnosing VWD Type 2A, 2B, or 2M (Flood et al. 2013).
Cons: VWD:CB assays typically use collagen type I or III. There is currently no common practice for assessing vWF binding defects to collagen types IV or VI. Therefore, analyzing vWF-platelet binding and standard VWF:CB binding assays may not be sufficient in assessing vWF functionality during increased bleeding (Keesler and Flood 2017).
FVIII binding assays
FVIII binding assays (VWF:FVIIIB) are recommended when diagnosing VWD Type 2N, particularly when genetic testing is unavailable (James et al. 2021). VWD Type 2N can be mistaken for hemophilia A due to low levels of FVIII. It is critical that the diagnose is accurate due to differences in treatments between VWD and hemophilia A. Hemophilia A is treated with FVIII concentrates, however due to vWF deficiencies in VWD Type 2N, additional FVIII proteins will not be sufficient to prevent bleeding. VWD Type 2N is best treated with vWF replacement therapy to prevent and treat serious bleeding (James et al. 2021). Genetic testing can identify known variants that cause VWD Type 2, however novel variants can be found, in which cases additional phenotypic analyses such as VWF:FVIIIB assays are required.
Pros: VWF:FVIIIB binding assays sensitively measure abnormal VWF-FVIII binding. Normal vWF will bind FVIII in this assay. VWD Type 2N samples will show defects in vWF-FVIII binding. VWF:FVIIIB binding assays are a critical step in distinguishing VWD Type 2N from hemophilia A.
The need for global hemostasis analysis during initial VWD screening
Individuals with borderline-low levels of vWF are challenging to diagnose. Low quantities of vWF in the blood does not guarantee that excessive bleeding will occur following injury. However, some patients with low levels of vWF do exhibit bleeding, which may be improved following treatment (Keesler and Flood 2017). Therefore, this presents the need for improved primary hemostasis assays that more accurately measure vWF functionality and platelet binding activities. The platelet function assays commonly used to investigate VWD are time-consuming and require large amounts of blood samples. Particularly when diagnosing VWD Type 1 or Type 2, there is a need for an assay to quickly and sensitively measure defects in primary hemostasis. A global primary hemostasis tool that measures disorders in platelet function would be valuable for use as a screening tool to aid in VWD diagnosis or prior to undergoing procedures that involve hemostatic challenges, such as surgery.
Additionally, a global primary hemostasis tool would prove useful in measuring drug responses during treatment. For example, it is recommended to administer desmopressin (DDAVP®) before using vWF blood products for VWD Type 1, 2A, 2M, and 2N. VWD subtype diagnosis needs to be accurate because DDAVP® causes thrombocytopenia in VWD Type 2B and Type 3. Instead, VWD Type 2B and Type 3 should be treated with VWF concentrate. In these instances, utilization of a global primary hemostasis tool would quickly identify the efficacy of DDAVP® treatment in promoting normal hemostasis. In instances where primary hemostasis is not improving, another therapeutic should be administered.
An example of a global primary hemostasis assay is the total thrombus formation analysis system (T-TAS®) 01. The T-TAS01 is an automated microchip flow-chamber system that utilizes capillary channels to monitor thrombus formation under blood flow conditions. No additional stimulation of platelets is necessary, unlike other platelet-binding activity assays. The area under the pressure-time curve (AUC) quantifies thrombus formation. This makes the T-TAS01 a valuable initial tool for evaluating platelet disfunctions.
A global hemostasis tool may bridge the gap between variable VWD defects and patient-specific bleeding risks in a technique that is quick and reliable.
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