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Published: July 2011Print Record of Viewing
Von Willebrand disease (VWD) is the most common of the inherited bleeding disorders. Plasma von Willebrand factor (VWF) multimer analysis is a qualitative visual assessment of the size spectrum and the banding pattern of VWF. This analysis, in conjunction with other tests of hemostasis, assists in the classification of von Willebrand disease.
This is the last in a four-part series on von Willebrand disease, and is also included in the series on test utilization.
Presenter: Rajiv K Pruthi, MBBS
Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.
Our presenter for this program is is Rajiv Pruthi, MBBS, Co-director of the Special Coagulation Laboratory and the Special Coagulation DNA Diagnostic Laboratory and Director of the Comprehensive Hemophilia Center at Mayo Clinic in Rochester, Minnesota. Dr. Pruthi will describe the role of von Willebrand factor multimer assay in the diagnosis of von Willebrand disease. After viewing the Hot Topic, we invite you to participate in Beyond Hot Topic. This question and answer session will be posted online approximately 1 month after the Hot Topic presentation is posted. You can submit a question for Dr. Pruthi using the information at the end of the presentation. Thank you, Dr. Pruthi.
The objectives of this presentation include a brief review of the biochemistry and physiology of von Willebrand factor, an overview of the laboratory procedure for plasma von Willebrand factor multimer analysis, and most importantly, to discuss the role of von Willebrand factor multimer analysis in the diagnosis of von Willebrand disease and in conclusion I will describe illustrative cases correlating abnormalities of von Willebrand factor multimer pattern with specific subtypes of von Willebrand disease.
We will start with a brief review of the biochemistry and physiology of von Willebrand factor.
Von Willebrand factor is a large glycoprotein that is synthesized in endothelial cells and megakaryocytes as individual units called monomers. Shown in this slide is a schematic of von Willebrand factor peptide above and below is shown the domain structure of a von Willebrand factor monomer. As you can see, each monomer has specific domains that serve different functions such as FVIII binding or binding to different receptors, such as the glycoprotein IIb/IIIa receptor on platelets.
Prior to secretion, these von Willebrand factor monomers undergo post-translational modifications such as glycosylation, dimerization and subsequently multimerization and propeptide cleavage ultimately resulting in secretion of von Willebrand factor peptides ranging in size from 0.6 to 20,000 kilodaltons. These are also categorized into low, intermediate, large and ultra-large molecular weight multimers.
Upon secretion, the ultra-large von Willebrand factor molecular weight multimer complexes undergo physiologic proteolysis by a mettaloprotease called ADAMTS-13. Thus, circulating plasma von Willebrand factor multimers reflect an equilibrium between secretion of von Willebrand factor containing large and ultra-large multimers and their cleavage into smaller derivatives which when visualized on agarose electrophoresis gels are categorized into low, intermediate and large or high molecular weight multimers.
We will next focus on the physiologic role of von Willebrand factor in primary hemostasis. As you can see from this slide, von Willebrand factor is not only present in plasma, but is also present in the subendothelial tissue and in platelets. Normally, platelets are circulating in an inactive state and are constantly bumping up against the vascular endothelium. Contact with an intact vascular endothelium does not result in platelet activation.
However, in the setting of a damaged vascular endothelium, subendothelial von Willebrand factor is exposed to circulating platelets, and binds to the constitutively activated glycoprotein Ib alpha platelet receptor. This binding action exerts a torque on the platelet, as it is briskly carried forward by flowing blood, and this results in platelet activation. Consequences include activation of multiple platelet receptors and secretion of intracellular contents which leads to recruitment of other platelets. All these actions culminate in platelet adhesion and aggregation resulting in formation of a platelet plug at sites of vascular injury. Simultaneous activation of the coagulation cascade results in formation of a stable hemostatic plug held in place by a fibrin clot. Thus, von Willebrand factor has an important role in platelet plug formation, in addition, von Willebrand factor acts as a carrier protein for FVIII, protecting it from proteolytic degradation.
We will now focus on the role of von Willebrand factor multimer analysis in diagnosis of von Willebrand disease.
I will use an illustrative case to discuss our approach to the evaluation of von Willebrand disease. A 23 year old female was referred for evaluation of a lifelong personal history of easy bruising and a recent dental extraction that was complicated by excessive bleeding. This, in conjunction with her maternal family history of bleeding prompted a referral for a bleeding evaluation.
When you see a patient with a bleeding disorder, there are a few disorders to keep in mind. As you can see, von Willebrand disease is the most common hereditary bleeding disorders across all ethnic groups. Among people of Ashkenazi Jewish background, factor XI deficiency is very common. This contrasts with the prevalence of the hemophilias.
At the Special Coagulation Laboratory at Mayo Clinic Rochester, we recommend an algorithmic approach to the diagnosis of von Willebrand disease. In this algorithm, called the von Willebrand disease Profile, initial assays for suspected von Willebrand disease include the von Willebrand factor antigen, activity and the FVIII activity. The von Willebrand factor multimer is only performed if indicated.
This next slide provides an overview of the von Willebrand disease profile. von Willebrand factor mutimers are performed only if indicated. So let us review what the indications for von Willebrand factor multimer analysis are. Reflexive von Willebrand factor multimer analysis is performed for plasma samples with a reduced von Willebrand factor antigen or activity, or a von Willebrand factor activity to antigen ratio below 0.8.
Coming back to our patient, as you can see, her von Willebrand factor antigen and activity were reduced as was the ristocetin cofactor activity, the latter assay is generally performed only for samples with reduced von Willebrand factor activity. The FVIII activity was normal, thus excluding a homozygous carrier state for von Willebrand disease Type 2 N or Normandy subtype. Based on these criteria outlined in the von Willebrand disease profile, von Willebrand factor multimer analysis was subsequently performed.
Let us address the question of why von Willebrand factor multimer analysis needs to be performed. As you can see, the main reason to perform von Willebrand factor multimer analysis is to sub-type von Willebrand disease. The main clinically relevant distinction is between type 1 von Willebrand disease and what I like to term the non-type 1 von Willebrand disease, that is the type 2 variants and type 3 von Willebrand disease. This distinction has therapeutic implications. For the type 1 von Willebrand disease, desmopressin or DDAVP, which may be administered by intravenous or intranasal route, may be an option for prevention or treatment of minor hemorrhage. For most type 2 variants and all patients with type 3 von Willebrand disease, von Willebrand factor containing clotting factor concentrates are the treatment of choice.
In addition to subtyping congenital von Willebrand disease, von Willebrand factor multimer analysis may be useful in certain acquired conditions. Some patients with aortic stenosis, hypertrophic obstructive cardiomyopathy, and in those with left ventricular assist devices experience bleeding, typically gastrointestinal bleeding. In this subset of patients, investigation into the von Willebrand factor multimer structure may provide clues to the cause of bleeding.
Next, we will briefly review the laboratory procedure for von Willebrand factor multimer analysis.
Broadly considered, the steps of von Willebrand factor multimer analysis consists of denaturation of plasma von Willebrand factor multimer with an anionic detergent, and then subjecting the plasma sample to agarose gel electrophoresis to separate the multimers. Generally, a discontinuous gel system helps in improving resolution, and this resolution will vary with the agarose gel concentration. At this stage, most laboratories electroblot the immobilized von Willebrand factor multimers from the agarose gel onto a membrane for subsequent labeling with an anti- von Willebrand factor antibody which may be radioactive or nonradioactive, chemiluminescent antibodies. At the Special Coagulation Laboratory, Mayo Clinic Rochester, our technique has always consisted of an in-gel analysis system which was originally radioactive, but more recently is an infrared based labeling system, thus avoiding radioactivity. The final step consists of a review of the multimers and interpretation taking into account the von Willebrand factor assay results.
Shown here is an example of our von Willebrand factor multimer gel plate. The discontinuous gel is poured in two steps and allowed to solidify.
After the gel solidifies, it is carefully removed from the plates and a template is placed on the gel to guide placement of the plasma sample wells as shown.
After the gel solidifies, it is carefully removed from the plates and a template is placed on the gel to guide placement of the plasma sample wells as shown.
The gels containing patient plasma samples are then subject to electrophoresis thus separating the von Willebrand factor multimers.
During electrophoresis, the smaller von Willebrand factor multimers move more rapidly through the gel than the larger multimers as shown on this slide.
After the electrophoresis is completed, the gels containing patient von Willebrand factor are subject to multiple incubation and washing steps as shown on the slide. As you can see on the right, our original radioactive method resulted in a processing time of approximately 7 to 9 days. With our developmental efforts, using an infrared-based system, processing time has been reduced to approximately 4 days, and has also eliminated the need for radioactive reagents.
Shown on this slide is an example of von Willebrand factor multimers using our original radioactive method. The gels were exposed to x-ray film and subsequently developed for interpretation.
With our current infrared method, the gels are scanned into an Odyssey scanning system and the images are digitally captured and archived.
The image generated by this method results in an infrared image, which is converted to a gray-scale image. These multimers may then be either visualized on a computer screen or printed for interpretation.
Shown on the next side are selected plasma von Willebrand factor multimer images from patients with von Willebrand disease. On the left are examples of images generated by our infrared method and on the right are images of the same plasma samples generated by the auto-radiographic method. The infra red image method provides superior resolution.
In summary, analysis of von Willebrand factor multimers is a labor-intensive process, and is not necessary in the initial laboratory testing for evaluation of von Willebrand disease.
In the subsequent slides I will provide illustrative examples of plasma von Willebrand factor multimer patterns seen in patients with specific von Willebrand disease subtypes.
Coming back to our case, this 23-year-old female had mildly reduced von Willebrand factor levels and her pattern of von Willebrand factor multimers demonstrated a normal distribution. She was thus diagnosed as having congenital type 1 von Willebrand disease. We performed a desmopressin trial and demonstrated that she responded very well therefore providing a useful option for prevention and treatment of minor hemorrhage. von Willebrand factor concentrates would still be required for prevention of bleeding during major surgeries and for treatment of major bleeding.
In the next case, this patient had markedly reduced von Willebrand factor levels. As a result of such marked reduction, factor VIII activity was also reduced. These data suggest either a severe type 1 or a type 3 von Willebrand disease. Analysis of this patient’s von Willebrand factor multimers essentially demonstrated absent von Willebrand factor multimers. Given the virtual absence of von Willebrand factor multimers, we do not attempt a desmopressin trial. Prevention and management of minor or major hemorrhage for patients with this type of von Willebrand disease would consist of von Willebrand factor concentrates.
This next patient had a von Willebrand factor activity and ristocetin cofactor activity that was disproportionately reduced compared to the von Willebrand factor antigen. Multimer analysis demonstrated a decrease in the higher molecular weight multimers consistent with type 2A von Willebrand disease.
The next patient also had a disproportionate reduction in von Willebrand factor activity assays compared to the von Willebrand factor antigen and there was an absence of the high molecular weight multimers; this patient was also thrombocytopenic and therefore fit the diagnostic criteria for type 2B von Willebrand disease. Patients with type 2 von Willebrand disease generally do not respond to desmopressin, in fact desmopressin is contraindicated in patients with type 2B von Willebrand disease. Management will therefore consist of use of von Willebrand factor concentrates for prevention and management of hemorrhage.
The next and final patient had a disproportionate reduction in the von Willebrand factor activity assay compared to the antigen. The multimer analysis however demonstrated presence of ultralarge molecular weight multimers. This patient had a rare variant called type 2M von Willebrand disease, due to an arginine to cysteine mutation in amino acid 1374. In this variant, desmopressin is not always effective and von Willebrand factor concentrates would be the mainstay of therapy.
In summary, von Willebrand factor multimerization is important for hemostasis. Analysis of von Willebrand factor multimers, however, is technically complex and time consuming. It’s main role is to subtype von Willebrand disease. It is not useful nor recommended for the initial screening for von Willebrand disease, nor is it useful for a condition called thrombotic thrombocytopenic purpura.
Von Willebrand factor multimer analysis should be performed only reflexively after initial screening tests such as von Willebrand factor antigen and activity are found to be reduced or have an abnormal ratio as outlined in the von Willebrand disease profile. There are certainly selected clinical conditions such as in patients with myeloproliferative disorders, hypertrophic obstructive cardiomyopathy or aortic stenosis where multimer analysis may be useful despite normal results of von Willebrand factor assays. However, the cutoffs for von Willebrand factor activity to antigen ratio validated during development of the von Willebrand disease profile have been set to be highly sensitive for detection of a acquired abnormalities of von Willebrand factor.
The final slide shows selected references that were used in preparation of this presentation and I thank you for your attention.