Laboratory Diagnosis of Inherited von Willebrand Disease

von Willebrand factor (VWF) is a large adhesive glycoprotein required for platelet adhesion to subendothelium at the site of vessel injury, platelet–platelet interaction to form the platelet plug, and stabilization of factor VIII (FVIII) in the circulation. Deficiency or defect of VWF leads to the common disorder of hemostasis, von Willebrand disease (VWD). Many cases of VWD, but not all, are due to defects in the structural gene for VWF, located on chromosome 12p. VWF forms a noncovalently bound complex with FVIII, protecting it from clearance. Because unbound FVIII has a shortened half-life, decrease in normal VWF may lead to a secondary decrease of FVIII coagulant activity (VIII:C), although the F8 gene on the X chromosome is normal.

The VWF gene includes 52 exons, covering 178 kilobases (kb) of DNA and encoding mRNA of about 9 kb. In addition, there is a pseudogene for VWF located on chromosome 22q, which is a copy of exons 23–34. The VWF gene produces a 2813-amino acid (AA) polypeptide (See Chapter 109 ). During processing in the endoplasmic reticulum, a 22-AA signal peptide is cleaved, dimers are formed by disulphide bonds at the carboxyl termini, and N-linked glycosylation occurs. In the Golgi, O-linked glycosylation occurs, large polymers called multimers are formed by disulphide bonds at the amino termini, and a 741-AA propolypeptide (VWFpp) is cleaved. The protein is secreted or stored as multimers reaching >20 million Daltons. After release from cells, the ultralarge multimers are reduced in size by proteolytic cleavage by the metalloproteinase ADAMTS13. Failure of cleavage results in the ultralarge multimers seen in thrombotic thrombocytopenic purpura. Normal VWF function requires the presence of high-molecular-weight (HMW) multimers.

Over 250 mutations in the VWF gene have been shown to cause VWD. They are listed in the International Society on Thrombosis and Haemostasis VWF database at http://vwf.group.shef.ac.uk . Many mutations are unique to individual families, while a few recur.

VWD is diagnosed and classified based on quantitative and qualitative differences in VWF. Type 1 VWD is a partial quantitative deficiency of VWF. Type 2 VWD results from a qualitative defect in VWF. Type 3 is a complete or nearly complete deficiency of VWF.

An algorithm for VWD testing is shown in Fig. 134.1 . Routine screening tests are not useful for most forms of VWD. The activated partial thromboplastin time is prolonged only in the minority of VWD patients who have significantly reduced VIII:C. Prothrombin time, thrombin time, and fibrinogen are normal. The platelet function analyzer will detect moderate to severe VWD, but it is not sufficiently sensitive to allow exclusion of the diagnosis. Accurate diagnosis and characterization of VWD subtype require a panel of tests measuring different aspects of the FVIII/VWF complex. Most commonly, VWF antigen (VWF:Ag), VWF activity, and FVIII activity are used for the initial diagnosis. A decrease in any of these tests warrants further investigation. The ratio of results on these tests may be used to choose additional tests to be performed, which may be available only in reference laboratories. The more specialized tests are used to classify the type of VWD present.

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Laboratory diagnosis of von Willebrand disease. FVIII , factor VIII; RIPA , ristocetin-induced platelet aggregation; VWF , von Willebrand factor; FVIIIB , FVIII binding to VWF.

Ristocetin cofactor (VWF:RCo) has been the most widely used method for measuring the activity of VWF. Platelet aggregation or agglutination by VWF occurs in vitro only when the antibiotic ristocetin induces a conformational change in VWF allowing binding to GPIb, a change that is induced by shear stress in vivo. Single-nucleotide polymorphisms (SNPs) have been identified in VWF near the ristocetin binding site, which inhibit binding of ristocetin and give a falsely low measure of functional activity in tests using ristocetin. These SNPs are more common in African Americans and may account for the reduced ratio of VWF:RCo/VWF:Ag often seen in that group. Use of GPIb binding assays without ristocetin overcomes this effect.