Practical-Haemostasis.com

A Practical Guide to Laboratory Haemostasis

 

Von Willebrand Factor [VWF] Assays: Introduction



Introduction

Von Willebrand factor [VWF] is a glycoprotein of up to 20 x 106 Daltons [Da]. It is synthesised in:
1) The vascular endothelial cells where it is stored in the Weibel-Palade bodies prior to release and
2) The bone marrow megakaryocyte and so is present in platelets.
VWF is initially synthesised as a series of very large multimers that are degraded by the metalloprotease ADAMTS13 (A Disintegrin-like And Metalloprotease domain with Thrombospondin type I motifs). A deficiency of ADAMTS13 leads to the accumulation of ultra high molecular weight VWF multimers and the clinical and laboratory features of thrombotic thrombocytopenic purpura (TTP). Assays for ADAMTS13 are covered elsewhere.
VWF has two primary roles:
- As a carrier protein for FVIII preventing its proteolytic degradation in plasma
- As an adhesive protein involved in the interaction between platelets and the blood vessel wall.

The gene for VWF spans approximately 172kb of genomic sequence, consists of 52 exons, maps to chromosome 12 [12p13.3] and encodes a protein of 2813 amino acids that includes a signal peptide of 22 amino acids, a prepropeptide of 741 amino acids [residues 23-763] and a mature protein of 2050 amino acids [residues 764-2800]. The mature protein is divided into a series of domains that contain the various functional domains - see Reference 1. The mature VWF protein found in the plasma has a half-life of approximately 12 hours (range 9–15 hours).
An unprocessed partial VWF pseudogene [exons 23-34] exists on chromosome 22.


Click HERE to load an expanded view of the above image.

Von Willebrand Factor: Summary of Abbreviations

Designation Summary
von Willebrand Disease [VWD] The disorder due to a deficiency or functional abnormality of von Willebrand Factor
von Willebrand Factor [vWF] Multimeric glycoprotein that acts both as a carrier protein for FVIII and as an adhesive protein in the platelet-vessel wall interactions
von Willebrand Factor Ristocetin Cofactor Activity [VWF:RCo] A functional assay of plasma VWF based upon the degree of platelet agglutination induced after the addition of ristocetin
von Willebrand Collagen Binding Activity [VWF:CB] A functional assay of plasma VWF that quantifies the ability of VWF to bind collagen-coated ELISA plates
Von Willebrand Activity [VWF:Act] A functional assay of plasma VWF that uses a monoclonal antibody that targets the part of the VWF molecule that binds to the GpIb receptor.
von Willebrand Factor Antigen [VWF:Ag] An immunological assay that quantifies the amount rather than the function of VWF in plasma
von Willebrand Factor Propeptide [VWFpp] The propeptide of von Willebrand Factor
FVIII A clotting factor that acts as a cofactor in the formation of the Xase ['Tenase'] complex
FVIII:C A functional assay of FVIII coagulant activity
FVIII:Ag An immunological assay of FVIII
Ristocetin-Induced Platelet Aggregation [RIPA] A test that measures the ability of VWF to bind to platelets
VWF gene The gene encoding von Willebrand Factor
F8 gene The gene encoding FVIII


The Diagnosis of Von Willebrand Disease [VWD]

Von Willebrand Factor assays are covered elsewhere in Practical-Haemostasis.com.
For more information on the diagnosis of VWD - see References 1-3.

Von Willebrand Disease [VWD] is classified into Types 1 - 3. Click HERE to load an Excel table which summarises the various VWD subtypes.

Tests Summary
Clinical evaluation of bleeding symptoms This can be difficult as mild bleeding problems are common in healthy individuals. The use of a standardised bleeding score can be very useful see References.
Family History Establishing the presence of any significant family history suggestive of a bleeding disorder.
Initial laboratory tests - Full blood count including platelet count
- PT
- APTT
- Fibrinogen
- [PFA-100]
VWF:RCo to VWF:Ag ratio





VWF:CB to VWF:Ag ratio
The VWF:RCo to VWF:Ag ratio can aid in the diagnosis of types 2A, 2B, and 2M VWD and help differentiate them from type 1 VWD.
A VWF:RCo/VWF:Ag ratio of <0.7 suggests the presence of a dysfunctional VWF.

A similar approach has been proposed for the use of the VWF:CB/VWF:Ag ratio.
In Type 2A VWD, the ratio is usually low.
In Type 2B VWD, the VWF:RCo/VWF:Ag ratio is usually low but may be normal.
In type 2M VWD, the VWF:Ag concentration may be reduced or normal, but the VWF:RCo/VWF:Ag ratio will be <0.7. 
Specialised laboratory tests Factor VIII binding studies
These can be of value in the identification of 2N VWD although this is not a widely available test and VWF gene analysis can be equally useful.
Multimer Analysis VWF multimer analysis is a qualitative assay that depicts the variable concentrations of the different sized VWF multimers present in plasma.
Low Dose RIPA Low dose ristocetin-induced platelet agglutination can be of value in identifying patients with 2B VWD
Initial laboratory tests in possible VWD VWF:Ag VWF:Ag is an immunoassay that measures the total amount of VWF in plasma but provides no information on its function
VWF:Act VWF:Activity refers to a functional assay of plasma VWF that uses a monoclonal antibody that targets the part of the VWF molecule that binds to the GpIb receptor. Other functional VWF assays may be undertaken including VWF Collagen binding assays [VWF:CB] and VWF Ristocetin assays [VWF:RCo]
FVIII:C FVIII:C is a measure of the cofactor function of the clotting factor VIII usually measured in a 1-stage functional assay.
VWF:RCo to VWF:Ag ratio
The VWF:RCo to VWF:Ag ratio can aid in the diagnosis of types 2A, 2B, and 2M VWD and help differentiate them from type 1 VWD.
A VWF:RCo/VWF:Ag <0.7 has been used to establish the presence of a dysfunctional VWF.
VWF:CB to VWF:Ag ratio A similar approach has been suggested for the VWF:CB/VWF:Ag ratio.
In type 2A VWD, the ratio is usually low and in type 2B VWD, the VWF:RCo/VWF:Ag ratio is usually low but may be normal.

In type 2M VWD, the VWF:Ag concentration may be reduced or normal, but the VWF:RCo/VWF:Ag ratio will be <0.7. 
VWFpp to VWF:Ag ratio This is useful in studying patients with Type 1 VWD and in separating Type 1 patients with a normal VWF half-life from those with a shortened half-life e.g. Type 1C VWD [VWD Vicenza].
VWF Inhibitor assays Screening for VWF inhibitors by means of a conventional inhibition assay [a mix of normal and test plasma and then assaying VWF levels] is very difficult . A standard Bethesda-type inhibitor assay will frequently fail to identify a VWF inhibitor. ELISA assays have been published for looking for VWF inhibitors but again do not always identify antibodies.
The history can be very important in such cases as there is frequently no history suggestive of a bleeding disorder until very recently. The half-life of infused VWF is also decreased and establishing this can be very useful. The measurement of the VWFpp to VWF:Ag ratio an also be very useful. Some examples of acquired Von Willebrand Syndrome [AVWS] are included in the Data Interpretation section of P-H.com.


The algorithm below summarises one possible approach to the investigation of a patient with possible VWD:


Data Interpretation

Click HERE to go to the Data Interpretation Exercises