Von Willebrand Disease: Elucidating the Clinical Picture Through the Use of Molecular Methods

Transcription

1 Von Willebrand Disease: Elucidating the Clinical Picture Through the Use of Molecular Methods Elisa B. Cardenas, MS, MT(ASCP), Owatha L. Tatum, PhD, MP(ASCP) (Molecular Pathology, School of Allied Health Sciences, Texas Tech University, Lubbock, TX) DOI: /LMSNLQO70WGWF0UK CE Update Abstract Von Willebrand disease is the most common congenital bleeding disorder in humans. This bleeding disorder affects men and women equally and has a prevalence of approximately 1% to 2%. It results from a quantitative or qualitative abnormality in von Willebrand factor. After reading this paper, readers should be able to discuss the history, molecular biology, and diagnosis of von Willebrand disease; compare and contrast the clinical presentation and testing used in hemophilia A versus von Willebrand disease; outline the process of diagnosis of von Willebrand disease using molecular methods; discuss the various What is von Willebrand Disease? Von Willebrand disease (vwd) is the most common congenital bleeding disorder in humans. The disease is autosomally inherited. 1 This bleeding disorder affects men and women equally and has a prevalence of approximately 1% to 2% in the general population. It results from either a quantitative or qualitative defect in von Willebrand factor (vwf), which is an adhesive glycoprotein. 2,3 Adhesive glycoproteins are a complex of proteins that are essential for platelet adhesion and aggregation. When an endothelial injury occurs, platelets attach to these adhesive proteins and the pathway for a clot is set into play. 7,9,15,27,39-41 The deficiencies in vwf that occur in individuals with this disease are due to mutations in the von Willebrand factor gene and classification of the disease is based upon these many mutations. Von Willebrand factor is not directly involved in the pathway leading to formation of a clot, but it is sometimes confused with deficiencies in factor VIII when studies of coagulation are conducted in the clinical laboratory. Clinicians who observe patients with bleeding disorders must use the family history and laboratory studies, including coagulation studies, in order to provide an accurate diagnosis because this disorder can be confused with hemophila A in patients who present with extremely low levels of vwf. At the present time, there are many tools that clinicians can use to provide an accurate diagnosis if von Willebrand disease is suspected. These tools include a family history, laboratory screening tests such as bleeding time (although this laboratory testing method is considered historical to many hematologists and clinicians), platelet count, prothrombin time (PT), partial thromboplastin time (APTT), fibrinogen, platelet function analyzer-100 (PFA-100) (Dade-Behring, Margburg, Germany), and special coagulation studies such as von Willebrand factor antigen (vwf:ag), von Willebrand factor activity (either ristocetin cofactor or collagen binding activity), ristocetin induced platelet agglutination (vwd:ripa), and von Willebrand factor multimeric structure analysis done by agarose gel electorphoresis. Within the last decade, molecular diagnostic testing has been developed and can be used to detect and classify mutations associated with this condition. 6,18,33-37 Clinical presentation of this disease includes bleeding from mucous membranes and easy bruising. The gene for von Willebrand factor is located on chromosome 12 and a pseudogene is located on chromosome 22. Von Willebrand disease is currently classified into different types based on abnormalities in von Willebrand factor that include types 1, 2, and 3. Within the last half century, many aspects of the disease have been thoroughly researched and, as a result, individuals affected with the condition can manage the disease in an effective manner. treatments for patients diagnosed with von Willebrand disease; and differentiate types 1, 2, and 3 von Willebrand disease from one another. Molecular Diagnostics exam and corresponding answer form are located after this CE Update article on page 749. Most patients affected by the disease will present with altered function of von Willebrand factor in primary and secondary hemostasis. 7,42 Persons affected with this type of von Willebrand disease suffer from mild bleeding episodes and may experience more severe bleeding during surgical procedures. Characteristic symptoms of this condition include bleeding from mucous membranes and subcutaneous tissue, gastrointestinal bleeding, gum bleeding, postpartum hemorrhages, menorrhagia in women, and easy bruising. Less common symptoms may include muscle hematomas and deep vessel hemorrhages. 8,16,28-31,33 Hemophilia A presents with a similar, but distinct, set of clinical characteristics, including hemorrhage into deep structures like muscle and joint spaces and delayed bleeding episodes after surgical procedures and, in severe cases, spontaneous hemorrhages are common. Other symptoms of hemophilia A are excessive bleeding after dental extraction and procedures, hematuria, and gastrointestinal bleeding. Table 1 compares von Willebrand disease with hemophilia A. Pregnant women with the disease will tend to have elevated levels of von Willebrand factor and factor VIII during pregnancy but commonly have bleeding complications during delivery and will most likely suffer from heavy bleeding for an extended period of time after giving birth. Therapy for von Willebrand disease may consist of desmopressin acetate (DDAVP) and von Willebrand factorcontaining concentrates. It is important to note that the use of cryoprecipitate as a treatment is strictly historical as the amount of vwf in cryoprecipitate is not regulated. Treatment with factor VIII-containing concentrates is now preferred by physicians. 16,18,19,28-30,33-37 Desmopressin acetate is a synthetic hormone that, when taken, causes the body to stimulate production of more von Willebrand factor and factor VIII from endothelial storage sites into the bloodstream and is commonly taken in the form of an injection. This type of treatment works well in patients who are affected by type 1 von Willebrand disease and also in patients affected with some subtypes of type 2. Von Willebrand-factor-containing concentrate is given as a treatment for von Willebrand factor deficiency and factor VIII replacement. This labmedicine.com December 2008 j Volume 39 Number 12 j LABMEDICINE 743

2 Table 1_Comparison of von Willebrand Disease and Hemophila A. A Comparison of Factory Deficiency, Mode of Inheritance, Individuals Affected, Screening Tests, and Molecular Diagnostic Assays Available for Both Conditions 24,35,37 von Willebrand Disease Hemophila A Factor deficency von Willebrand factor Factor VIII Mode of inheritance Autosomal X-linked Those affected Both males and females Usually males, rarely females Bleeding time Prolonged Prolonged Prothrombin time (PT) Normal Normal Partial thromboplastin time (APTT) Prolonged Prolonged Specific assays used in lab diagnosis vwf antigen (vwf:ag) Factor VIII activity assay (VIII:C) Ristocetin cofactor assay (vwf:rcof) Factor VIII antigen assay (VIII:Ag) Collagen binding assay (vwf:cba) vwf multimer assay (vwf:multimer assay) Ristocetin induced platelet agglutination (vwf:ripa) Available molecular diagnostic testing Analysis of the entire coding region: sequence analysis Analysis of the entire coding region: sequence analysis Sequence analysis of select exons Sequence analysis of select exons Targeted mutation analysis Analysis of the entire coding region: mutation scanning Linkage analysis Targeted mutation analysis Prenatal diagnosis Linkage analysis Carrier testing X-chromosome inactivation study Deletion/duplication analysis Prenatal diagnosis Carrier testing type of treatment works well in patients that are affected with type 1 von Willebrand disease that are not responsive to DDAVP, type 2, or type 3, or in those patients that cannot be treated with DDAVP. Antifibrolytic drugs can also be used in conjunction with DDAVP and replacement therapy, but this type of therapy is usually used to stop bleeding after minor surgery, injury, or tooth extraction. Treatments for women with this condition who suffer from heavy menstrual bleeding may include using a combination of oral contraceptives, which can cause an increase in production of von Willebrand factor and factor VIII, and aminocaproic acid or tranexamic acid, which are antifibrinolytic drugs that can decrease bleeding by decelerating the breakdown of clots. With proper diagnosis and appropriate treatment, persons affected with von Willebrand disease can manage the disease and live active and productive lives. 20,21 History of von Willebrand Disease An internist in Helsinki, Finland, Erik A. von Willebrand first described a bleeding disorder in 1926 in a family from Foglo on the islands of Aland in the Gulf of Bothnia (between Finland and Sweden), in his original publication Hereitar pseudohemofili (Hereditary pseudohaemophilia). 4,5 The first case of the same type of bleeding condition was later observed in the United States in In 1950, the first clinical assay for factor VIII was developed and many physicians reported patients of both sexes with prolonged bleeding times associated with deficiencies in factor VIII. In 1971, it was discovered that another immunological factor was responsible for this disease, called von Willebrand s factor, that was different from factor VIII deficiency and resulted in its differentiation from hemophilia A. 4,6 In the years to follow (early- to mid-1970s), von Willebrand antigen, ristocetin-induced platelet aggregation, and ristocetin cofactor activity were discovered and led to the development of assays that in turn led investigators in the late 1970s to identify different types of von Willebrand disease. In the early 1980s, the first large population epidemiological studies were published and revealed the actual frequency, found to be high, of von Willebrand disease deficiencies. In 1985, the von Willebrand factor gene was identified and cloned by 4 individual groups and resulted in a better understanding of von Willebrand factor and of the resulting disease on a molecular level. Other important milestones include the development of a vwf-binding assay to collagen in 1986 and a vwf-binding assay to FVIII and vwd 2N variant in In 1994, classification of von Willebrand disease distinct types was accomplished and in 1998 to 2002 national guidelines for the clinical diagnosis of the disease were established. In 2002 to 2006, a standardized bleeding score for von Willebrand disease was introduced for clinical use. 5 What is von Willebrand factor? Von Willebrand factor (vwf) is produced in megakaryocytes and endothelial cells and undergoes further processing (dimerization and polymerization) to form very large molecular weight multimers. Dimer assembly takes place early during the processing of the protein by configuration of disulfide bonds between the C-terminal ends of the pro-vwf, which occurs in the endoplasmic reticulum (ER), and the addition of N-linked carbohydrate residue, which also takes place in the ER, is a very important procedure involved in the formation of dimers and exit from this area. The pro-vwf dimers are carried to the Golgi apparatus, where further glycosylation and sulfation happens and multimers are formed by disulfide bonds arranging between the N-termini of the dimers. It is first synthesized as a pre-pro molecule consisting of a 22-amino acid signal peptide that is then followed by a propeptide consisting of 741 amino acids. The mature vwf protein consists of 2050 amino acids. 9,32 Von Willebrand factor peptide is composed of homologous domains which consist of 3 A domains, 3 B domains, 2 C domains, and 4 D domains, and each domain has unique functional properties. The larger multimers of vwf are stored in the Weible-Palade bodies of endothelial cells, and the alpha granules of platelets serve as a storage site for the factor as well. It is a glycoprotein that circulates in the plasma and its role in primary hemostasis is to carry/ stabilize factor VIII and to act as a gummy link between platelets 744 LABMEDICINE j Volume 39 Number 12 j December 2008 labmedicine.com

3 Figure 1_Platelet adhesion and the role of von Willebrand factor. Platelets interact with von Willebrand factor in the subendothelium leading to eventual clot formation. and vascular subendothelial structures as represented in Figure 1. The fastening of vwf to platelets and subendothelial structures is vital for conventional platelet adhesion and for the platelet aggregation that occurs at high shear rates. Fastening to platelets requires preliminary activation and modification in the configuration of vwf so that the binding sites in the A1 domain can link the platelet receptor Gp Ib-IX-V complex on the platelet surface, which occurs on platelets that are active and inactive. A second platelet receptor for vwf, known as Gp IIa/IIIa, does not link the platelets unless they are stimulated/activated. When the platelets are activated, Gp IIa/IIIa endures conformational alteration and becomes available on the platelet surface, which can be brought on by an array of factors including activation provoked by Gp Ib binding to immobilized vwf. Interaction amid platelets, Gp IIb/ IIIa, and vwf seems to be a factor in the absolute, nonreversible linking of platelets to the subendothelium after vwf has attached to Gp Ib and may also contribute to platelet aggregation, which is especially true under high shear conditions. Under low shear conditions, platelet aggregation is interceded primarily by Gp IIb/IIIa binding to fibrinogen. The vwf also binds to numerous forms of collegen (types I, II, III, IV, V, and VI) but the exact components in the subendothelial connective tissue that bind specifically are not yet well understood. It is important to note that type VI collagen is especially important to this process. Its function in secondary hemostasis is to unite with factor VIII, which would otherwise have a very short half-life, and carry/secure it in the plasma. The vwf generally guards factor VIII from proteolytic inactivation by activated protein C and its cofactor protein S. Without vwf, the half-life of factor VIII in the circulation is estimated to be 2 hours and the inactivation of factor VIII by activated protein C is reduced in speed by almost 10- to 20-fold when vwf is present. It is essential to note that vwf can only bind with factor VIII when it has not been cleaved by thrombin. 6,7,10,16,28,29 Synthesis of the factor is partially regulated by hormones such as estrogen and thyroid hormones. The normal levels of vwf in the plasma are about 500 to 1,000 µg/ dl but levels fluctuate widely from individual to individual and the concentrations are also influenced by the ABO blood groups. Normal individuals with blood type O have about 30% lower levels of vwf than individuals with blood types A, B, or AB, but the reason for this is unknown.when vwf binds with factor VIII, it enhances the half-life 5-fold (the usual half-life of vwf is 8 to 12 hours). 6,9 Figure 2_von Willebrand factor gene, pseudogene, and corresponding mrna transcript. Depiction of the domains in the von Willebrand factor protein and associated functions of each domain. labmedicine.com December 2008 j Volume 39 Number 12 j LABMEDICINE 745

4 Molecular Biology of von Willebrand Disease The gene that encodes for vwf is positioned on the short arm of chromosome 12 spanning 180 kb and contains 52 exons. The gene is transcribed to yield an mrna fragment of almost 9 kb. It is important to note that there is also a pseudogene located on chromosome 22 (22q11-q13) that represents a copy of the genomic DNA from exons of the vwf gene, making molecular studies of vwf a bit tricky (Figure 2). Special measures to ensure elimination of the pseudogene must be taken when conducting genetic testing for vwf gene mutations because, if not, a false-positive or false-negative may result. The structure of the vwf gene and associated pseudogene are illustrated in Figure 2. The vwf gene is very large at 178 kb resulting in 52 exons. As would be expected with a gene of this size, it contains a number of alu and simple sequence repeats. Its size also points to a mechanism of origin from a segmental duplication. The 21- to 29-kb-long pseudogene region on chromosome 22 corresponds to exons 23 and 34 of the vwf gene on chromosome 12. Measures that can be used in molecular genetic testing may include restriction enzyme digestion or specific primer design to eliminate the pseudogene prior to polymerase chain reaction (PCR) amplification. 10,22,38 When an individual has von Willebrand disease, mutations are present in the vwf gene that cause either qualitative or quantitative defects in the factor. In most cases, qualitative defects are the products of missense mutations and quantitative Table 2_von Willebrand Factor Protein Domains Domains Interactions Table 3_Genetic Characteristics and Resulting Defects in vwd Types defects are products of large deletions, promoter, and frameshift nonsense mutations. 11 The vwf gene encodes a protein with numerous copies of homologous motifs consisting of 3 A, 3 B, 2 C, and 4 D motifs. The motifs encode for protein domains that provide the various functions of vwf. The A1 domain of this protein contains binding sites for platelet Gp Ib and ristocetin, the A2 domain contains a protease-sensitive domain that investigators think may play a part in regulation of vwf. The A3 domain is believed to have a focal collagen binding domain. The C1 domain has an RGD sequence that is a primary adhesive motif containing the amino acids Arg-Gly-Asp, and is thought to be capable of interacting with platelet Gp IIb/IIIa. Finally, D1, D, and D3 domains have been found to contain a factor VIII binding sequence. 9,12,23,32,38 The interactions of each domain are summarized in Table 2. Classification of vwd Types and Subtypes Currently, von Willebrand disease is classified into various types based upon qualitative and quantitative abnormalities in von Willebrand factor that include types 1, 2, and 3. The genetic characteristics and resulting defects for each type are summarized in Table 3. Type 2 is subdivided into subgroups A, B, M, and N. 13 Type 1 vwd is the most familiar, traditional form (80% of observed cases) and results from a fractional decrease in all Encoded Protein Motifs A A1 Contains binding sites for platelet Gp Ib and ristocetin A2 A2 domain contains a protease-sensitive domain which investigators think may play a part in regulation of vwf A3 A3 domain is believed to have a focal collagen binding domain B C C1 C1 domain has an RGD sequence that is a primary adhesive motif containing the amino acids Arg-Gly-Asp, and is thought to be capable of interacting with platelet Gp IIb/IIIa. D D domain has been found to contain a factor VIII binding sequence D D1 D1 domain has been found to contain a factor VIII binding sequence D3 D3 domain has been found to contain a factor VIII binding sequence Known Mutations vwd Type Associated Exon Codon Defect Inheritance Pattern Type 1 vwd 15 mutations 26, 28, Fractional decrease of all the sizes of Autosomal dominant von Willebrand factor molecular weight multimers Type 2A vwd 71 mutations 12, 13, 14, , 771, , Loss of high molecular weight multimers of Autosomal dominant 15, , 2773fs, vwf and decreased platelet-dependent function Type 2B vwd 52 mutations Loss of high molecular weight multimers of Autosomal dominant vwf resulting from an increase affinity for platelet glycoprotein 1b Type 2M vwd 18 mutations 18, 27, , 1205, Reduced interaction of vwf with platelets Autosomal dominant not connected due to a lack of high molecular weight multimers Type 2N vwd 37 mutations Reduced interaction of vwf with factor VIII Autosomal dominant resulting in significantly low levels of factor VIII Type 3 vwd mutations throughout Complete absence of synthesis of the vwf Autosomal recessive the vwf gene 746 LABMEDICINE j Volume 39 Number 12 j December 2008 labmedicine.com

5 the sizes of von Willebrand factor molecular weight multimers. Persons affected with type 1 vwd present clinically with mild clinical symptoms, and in some cases persons without a family history of bleeding may be asymptomatic. Type 2 von Willebrand disease results from an absence of high molecular weight multimers and persons affected typically have mild to moderate clinical symptoms. Type 3 von Willebrand disease is due to the complete absence of synthesis of the vwf and is considered to be the most rare and severe form. Patients affected with type 3 von Willebrand disease have severe clinical symptoms including severe bleeding from mucosal hemorrhages and muscle hematomas similar to hemophilia A but differ in that the bleeding time in these individuals is extremely prolonged. 8,9,18,32-34 Molecular Diagnosis of von Willebrand Disease With the identification of the von Willebrand factor gene in 1985 came the ability to explore and manipulate genomic DNA of persons affected with von Willebrand disease in order to screen for mutations associated with the condition. As stated above, when conducting molecular testing of mutations associated with the condition, the von Willebrand factor pseudogene located on chromosome 22 must be eliminated. One of the ways that the pseudogene can be eliminated is by digesting the genomic DNA that is to be screened with a restriction enzyme such as NcoI. The von Willebrand pseudogene contains an NcoI cutting site and digesting the genomic DNA with this restriction enzyme prior to PCR amplification will destroy it and therefore coamplification of the pseudogene will be avoided. 26 This is illustrated in Figure 2. Current testing methods used in the clinical laboratory available for screening of mutations associated with von Willebrand disease include sequence analysis of the entire coding region of the gene, sequence analysis of select exons, targeted mutation analysis, and linkage analysis. Prenatal diagnosis and carrier tests are also available. 24 Sequence analysis of the entire coding region is a procedure in which the nucleotide sequence is determined for the complete coding region of a gene. In sequence analysis, the actual nucleic acid sequence of a DNA fragment is determined and compared with a reference sequence. Sequence analysis of a select exon is a method of testing that consists of specific exons sequencing to identify sequence alterations and this method is used when certain exons are thought to contain the mutation that causes the targeted disease. Targeted mutation analysis is a method that tests for a nucleotide repeat expansion on one or more particular mutations associated with a disease. Linkage analysis, also known as indirect DNA analysis, is a method that tests for polymorphisms in a DNA sequence that are within the gene or close to the gene to track the inheritance of a disease causing mutation of that specific gene. Prenatal diagnosis (prenatal testing on a fetus) can be conducted during pregnancy to determine whether a fetus will be affected with a given disease. Several techniques, such as chorionic villus sampling, early amniocentesis, amniocentesis, and placental biopsy, may be used to obtain a specimen from the fetus or access fetal anatomy. Carrier testing (heterozygote testing or carrier detection) may be used to determine if an individual who may be asymptomatic possesses a genetic mutation that may cause a disorder that is inherited in an autosomal recessive or X-linked manner. 24,25 Future Clinical Directions and Concluding Remarks Von Willebrand disease is a bleeding disorder that affects about 1% of the population and does not seem to exhibit an ethnic bias. Within the last half century, many important aspects of the disease have been thoroughly examined in both basic biomedical research and in the clinical setting. As a result, individuals affected with this condition can now manage the disease in an effective manner, enabling them to live full and productive lives. 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