Von Willebrand factor and von Willebrand's disease: a complex protein and a complex disease

Von Willebrand factor is a complex protein which is important in several ways for normal hemostasis. Von Willebrand's disease results when there is either a quantitative or qualitative disorder of von Willebrand factor. In this review, the structure and function of von Willebrand factor are discussed. Additionally, the current laboratory and clinical classification of von Willebrand's disease and closely related variants are outlined.     

Endothelial cell surface expression and binding of factor VIII/von Willebrand factor.

Factor VIII/von Willebrand factor (FVIII/vWF), a glycoprotein molecular complex found in human plasma, has been demonstrated by cell membrane fluorescence to be present on the surface of cultured human umbilical cord vein endothelial cells. The endothelial nature of these cells was established by electron-microscopic studies that revealed the presence of Weibel-Palade bodies in virtually all cells cultured. A newly developed radioreceptor assay was used to detect FVIII/vWF in the medium taken from these endothelial cell cultures; FVIII/vWF concentration in the medium samples increased with time in culture. FVIII/vWF binding studies showed no significant FVIII/vWF-specific binding to endothelial cell surfaces and did not corroborate a previous report suggesting a FVIII/vWF-specific receptor on human umbilical cord vein endothelium. The presence of FVIII/vWF on endothelial cell membranes and the lack of receptor-mediated binding suggests that the FVIII/vWF either has been absorbed non-specifically to the cell surface or is an integral part of the endothelial cell membrane.     

Von Willebrand factor and platelet adhesiveness

A modification of Salzman's method has been used in an attempt to provide an assay in vitro for the von Willebrand factor. Platelet adhesiveness was increased in von Willebrand's disease by previously coating the beads with normal or haemophilic plasma or cryoprecipitate, whereas von Willebrand plasma had no corrective effect. Antihaemophilic factor (AHF) concentrates were studied in the same way and results compared with experiments in vivo.     

Detection of factor VIII von Willebrand factor in endothelial cells in first-trimester fetuses

Factor VIII von Willebrand factor was studied by the immunoperoxidase method in 38 cases of first-trimester therapeutic abortion and two cases of early second-trimester therapeutic abortion. Positive immunostaining was observed in endothelial cells at all gestational ages studied. The findings demonstrate the presence of factor VIII von Willebrand factor in endothelial cells as early as four weeks' gestational age.     

Von Willebrand's disease, hemophilia A, and factor VIII

Von Willebrand's disease is a bleeding disorder due to the combination of both a missing plasma factor and a deficiency of antihemophilic factor. The low levels of antihemophilic factor in von willebrand's disease have led many investigators to confuse it with or compare it to hemophilia A. It will become clear in this discussion that these two diseases differ clinically, genetically, and immunologically. Comparative studies of these diseases have helped elucidate the mechanisms of factor VIII biosynthesis in normal and abnormal states. A comprehensive model is presented.     

Von Willebrand factor,ADAMTS13, and thrombotic thrombocytopenic purpura

Von Willebrand factor (vWF), a glycoprotein critical for supporting platelet adhesion and aggregation at sites of vessel injury, exists in the plasma as a series of multimers. Recent studies have shown that a metalloprotease cleaves endothelial vWF to a series of multimers. A deficiency of the protease activity due to autoimmune IgG inhibitors or genetic mutations is associated with thrombotic thrombocytopenic purpura (TTP). Positional cloning based on kindreds with a genetic deficiency of the protease and amino acid sequencing of the purified protein have identified the protease as a novel member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 repeat) zinc metalloprotease family located on the long arm of chromosome 9. Mutations of the gene are detected in patients with the congenital form of TTP. These findings support the view that vWF proteolysis is critical in regulating vWF-platelet interaction and set the stage for improving the diagnosis and treatment of thrombotic thrombocytopenic purpura.     

Next-generation sequencing of von Willebrand factor and coagulation factor VIII genes: a cross-sectional study in Croatian adult patients diagnosed with von Willebrand disease

AimTo identify the von Willebrand factor (VWF) gene variant status in Croatian adult patients diagnosed with von Willebrand disease (VWD), provide differential diagnosis of VWD subtypes, and identify patients with mild hemophilia A (HA) who were earlier misdiagnosed as VWD.MethodsCoagulation testing included determination of VWF gain-of-function mutant glycoprotein Ib binding activity (VWF:GPIbM), VWF antigen, VWF collagen-binding activity, and multimeric analysis. Genetic analysis of VWF and FVIII genes was performed with next-generation sequencing (NGS).ResultsThe study enrolled 50 patients (72% women; median age 37 years, range 18-75) from 44 unrelated families. Fourteen patients were heterozygous for VWF gene variants compatible with type-1 VWD. Twelve had variants associated with type 2, of whom seven were classified as type 2A, four as type 2B, and one as type 2N. Six type-3 VWD patients were either homozygotes for null variants or combined heterozygotes. Eleven variants within the VWF gene were novel. Three female patients had variants within the FVIII gene, and were re-classified as mild-HA carriers, of whom one had causative novel variants both within VWF and FVIII genes. Fifteen patients remained without a defined genetic cause of their disorder, of whom five had VWF:GPIbM levels below 50%.ConclusionCroatian adult patients with VWD have considerable genetic heterogeneity. NGS of both VWF and FVIII genes provided accurate differential diagnosis of VWD subtypes and distinction of VWD from mild HA.

Von Willebrand disease (VWD) is the most frequent autosomally inherited bleeding disorder caused by quantitative deficiency or qualitative changes in the von Willebrand factor (VWF) protein structure. The prevalence of symptomatic cases among the general population ranges from 0.01% to 1%. This large multimeric plasma glycoprotein mediates platelet adhesion and aggregation at the vascular injury site through interaction with the platelet glycoprotein Ib (GPIb) and collagen in the subendothelium. Additionally, it transports and stabilizes coagulation factor VIII (FVIII) in circulation (1-3). Multiple VWF functions in hemostasis are regulated by different regions within the encoding gene, which is located on chromosome 12 and comprises 52 exons. To date, more than 700 unique disease-associated variants have been discovered throughout the whole VWF gene, affecting either VWF assembly, secretion, proteolysis, clearance, and/or binding affinity to GPIb, collagen, and FVIII (1,4). Any impairment of VWF function results in bleeding phenotypes, whose severity depends on the causative pathophysiological mechanism and the remaining levels of functional VWF (2). The majority of patients present with mild to moderate mucocutaneous bleeding and prolonged bleeding after trauma (5). The most severe cases suffer from spontaneous bleeding episodes, including joint, muscle, and intracerebral hemorrhage (5). Up to 80% of all VWD cases have a certain quantitative deficiency of a functionally and structurally normal VWF, which is classified as type-1 VWD. Overall, 15%-20% of cases have qualitative disorders within VWF, classified as subtypes 2A, 2B, 2M, and 2N relative to the underlying structural and functional defect. Specifically, type 2A is characterized by selective loss of high (HMWM) and/or intermediate molecular weight multimers, which results in a decreased ability of VWF to bind GPIb and collagen. Disorder of VWF in type 2B is related to enhanced binding of VWF to GPIb, thus causing increased platelet clearance and loss of HMWM. Type 2M is caused by decreased binding of VWF to GPIb, and is usually associated with a normal multimeric profile, while type 2N results in decreased VWF binding to FVIII in circulation. Type-3 VWD, the most severe form, characterized by undetectably low VWF levels, occurs in approximately 1 in a million people (2,6). Importantly, about 35% of all patients with mildly decreased VWF levels lack distinctive genetic markers. This is considered a separate entity termed “low VWF” (7).Determination of VWF activity in terms of its capability to bind platelet GPIb, together with VWF antigen (VWF:Ag) levels and FVIII coagulant activity (FVIII:C), represents the mainstay of VWD laboratory diagnostics. VWF activity can be determined either with ristocetin cofactor activity assays that measure the ability of VWF to bind GPIb in the presence of ristocetin, or with immunoturbidimetric assays that use latex microparticles coated with GPIb containing two gain-of-function mutations that enhance VWF binding (VWF:GPIbM). Distinguishing different type-2 VWD subtypes reguires additional investigation of VWF structural and functional features by means of activity assays that measure VWF binding to collagen (VWF:CBA) as well as multimeric analysis (5). Proper VWD classification is the basis for adequate treatment and patient counseling (6). Given the multifunctional nature and structural complexity of VWF, heterogeneity of underlying genetic variants, and variable bleeding tendency, accurate differential diagnosis of VWD is often challenging. The exact nature of causative VWF defect might remain incompletely revealed when commonly available laboratory assays are used (6,8). Patients presenting with ambiguous mild-bleeding phenotypes coupled with borderline levels of VWF (30%-50%) are especially hard to be properly characterized (7). Difficulties may also arise due to similarities in clinical presentation and laboratory results to patients with the mild form of hemophilia A (HA), especially in type 2N VWD characterized by similarly low FVIII levels. HA is a well-known inherited bleeding disorder characterized by decreased FVIII levels (9-12).Simultaneous genetic analysis of both VWF and FVIII genes with next-generation sequencing (NGS) allows a definitive differential diagnosis of VWD subtypes and possible distinction from mild HA. It also reveals the inheritance risk for family members. This approach enabled unambiguous diagnosis of either VWD or mild HA in patients with mild bleeding disorders (10,11). In addition, NGS of the VWF gene in Spanish patients resulted in reclassification to a different VWD subtype in approximately one quarter of participants (13). Each study conducted so far identified a subset of novel, disease-associated variants, confirming the complexity of the underlying genetic basis of VWD and further clarifying heterogeneous clinical presentation (13-17). The presence of compound heterozygosity in 37.5%-63.3% of patients additionally contributes to disease heterogeneity (14-16). Furthermore, distribution and frequency of variants within the VWF differ relative to geographic localization and ethnicity (13-15,17). Given the lack of such studies in Croatia, the genetic basis of VWD among Croatian population remains unknown.The aim of the present study was to provide molecular diagnosis by means of NGS in Croatian adult patients diagnosed with VWD, and thereby reveal the VWF gene variant status and associated VWD subtypes. Additionally, by simultaneous analysis of both VWF and FVIII genes we aimed to identify patients with mild HA who were earlier misdiagnosed as VWD.     

Von Willebrand factor purification from human plasma cryoprecipitate

Summary Von Willebrand factor (vWF) and fibronectin (Fn), as a by-product, were prepared from human plasma cryoprecipitate by a three-step chromatographic procedure. The respective final products were concentrated (80 U RCo/ml and 5 g/l, respectively), highly purified (specific activity >180 U RCo/mg protein and >95%, respectively) and biologically active.     

Platelets, von Willebrand factor, and prostaglandin I2

Depending on the time of addition, prostaglandin I2 (PGI2; greater than or equal to 10(-9) M) either inhibits or reverses platelet agglutination mediated by human factor VIII-related von Willebrand factor activity (FVIIIvWF) and ristocetin, or bovine FVIIIvWF alone. 6-Keto-PGF1 alpha, the inactive metabolite of PGI2, is without effect, PGI2 inhibition is potentiated by the phosphodiesterase inhibitor, theophylline, and is not the result of PGI2 suppression of ADP release. PGI2 (+/- theophylline) does not inhibit ristocetin-induced binding of purified human 125I-FVIIIvWF multimers to washed platelets or to platelets treated with PGI2 and then formalin fixed (although subsequent agglutination of these platelets is impaired). Washed platelets treated previously with 2-aminoethylisothiouronium bromide (AET), an agent that reduces disulfide bonds and alters platelet membranes, also bind human 125I-FVIIIvWF multimers without agglutinating. We conclude that FVIIIvWF-mediated agglutination requires both functional platelet FVIIIvWF binding sites and platelet-platelet cohesion sites, and that platelet surface cohesion sites are altered by AET and PGI2. PGI2 from adjacent intact endothelial cells may prevent excessive platelet accumulation on exposed subendothelium without suppressing an essential hemostatic process--the binding of platelets to subendothelial FVIIIvWF.     

Detection of von Willebrand disorder and identification of qualitative von Willebrand factor defects. Direct comparison of commercial ELISA-based von Willebrand factor activity options

Two von Willebrand factor (vWF):collagen binding (activity) assay (CBA) kit methods are commercially available. A monoclonal antibody (MAB)-based enzyme-linked immunosorbent assay (ELISA) system reported to correlate with a standard vWF:ristocetin cofactor (RCof) assay is also commercially available. It is marketed as a vWF:Activity assay and is available in 2 assay version formats. In the present study, these 4 vWF-activity options were compared directly with in-house vWF:CBA ELISAs for their ability to detect von Willebrand disease (vWD) and identify qualitative vWF defects. The 2 MAB-based systems detected vWD but could not specifically identify qualitative vWF defects, although the recently modified Mark II kit was more effective for the latter compared with the original Mark I kit. All vWF:CBA methods, including in-house and commercial, also effectively detected vWD but differed in their ability to identify qualitative vWF defects. Effectiveness was highest using the in-house reference vWF:CBA (using a type I/III collagen mix product from equine tendon), the Gradipore vWF:CBA (also uses equine tendon-derived collagen), or the in-house vWF:CBA methods using type III human collagen at a relatively low concentration (1 or 3 micrograms/mL, without covalent linkage). The IMMUNO vWF:CBA seemed to be the least effective among the vWF:CBA methods for detection of qualitative vWF defects.