von Willebrand factor is a large glycoprotein, composed of a series of protein polymers (multimers), which consist of repeating subunits linked by disulfide bonds. The number of subunits in each multimer varies, imparting a range of molecular weights to the multimers. The multimeric structure of vWf is important as the higher molecular weight multimers are more effective in hemostasis. von Willebrand factor can be measured in a variety of ways, including measurement of antigen, qualitative multimeric analysis, functional and genetic assays.
Antigen measurement
The amount of vWf can be measured in plasma using rocket immunoelectrophoresis (Laurell immunoassay) or enzyme-linked immunosorbent assays (ELISA). Both techniques are immunologic and detect vWf using anti-vWf antibodies. The vWf measured in this way is called von Willebrand factor antigen (vWf:Ag). This is the most commonly used method (especially using ELISAs) to quantitate vWf and is used by the Comparative Coagulation Laboratory at Cornell University. In these methods, the amount of vWf:Ag in the patient plasma is compared to that in a standard species-specific plasma pool, designated with a vWf:Ag value of 100%. For information on test interpretation, see related links or vWD page.
Multimeric analysis
Qualitative assays for vWf involve evaluation of the multimeric structure of vWf. This used to be done using crossed immunoelectrophoresis, but has been replaced by multimeric assay, in which the individual multimers comprising vWf are visualized on a gel. Multimeric analysis is important for distinguishing between types of von Willebrand disease, particularly type II vWD, in which vWf:Ag may be normal or reduced, but there is a relative decrease in the number of high molecular weight multimers.
Functional assays
Aggregation
The functional activity of vWf can be measured in vivo using the Buccal mucosal bleeding time (BMBT), and in vitro using a variety of assays. The most common assays for measurement of vWf function are those based on platelet aggregation, using ristocetin and botrocetin as agonists. The aggregation induced by these agonists is dependent on vWf, hence abnormal platelet aggregation indicates a deficiency or abnormality of vWf, or the platelet receptors (glycoproteins Ib-IX and IIb/IIIa) that bind vWf. These assays are only performed by veterinary laboratories that specialize in vWf testing.
Collagen binding assay
An additional in vitro functional vWf assay is the collagen binding assay. In this assay, vWf is evaluated for its ability to bind to a collagen surface in an ELISA assay. In human patients, the collagen-binding ability of vWf correlates well to platelet aggregation assays. This assay has been evaluated in the dog, and appears to be promising based on preliminary results.
Genetic assays
The underlying genetic defect in a variety of dog breeds, including the Doberman, Shetland Sheepdog, Manchester Terrier and Scottish Terrier, has been purportedly identified by a Michigan-based genetic company (VetGen). The accuracy of their genetic tests has not been validated by independent researchers and their results have not been published in peer-reviewed journals. Nevertheless, based on preliminary comparisons between vWf:Ag results from Cornell University and genetic test results from VetGen, the genetic test for the Scottish Terrier appears to be quite accurate. There have been some discrepancies in the Doberman with the genetic test, which are of concern. At this stage, genetic testing is only recommended for detection of carriers of genetic defects in vWf. The genetic test does not correlate to potential for bleeding (whereas there is some correlation between vWf:Ag results and potential to bleed in the Doberman breed) and does not aid in the diagnosis of severe type III vWD in dogs, which are readily identified by vWf:Ag testing.
Related links
- Comparative Coagulation Laboratory at Cornell University: Testing and information on vWD.
