Biosynthesis of human von Willebrand factor

Endothelium forms the inner lining of all blood vessels and, as a consequence, is in direct contact with the blood. Because of this and the synthesis and secretion of hemostatic components, the endothelium is able to modulate coagulation and fibrinolysis. An important hemostatic factor synthesized by endothelial cells is the von Willebrand factor (vWF). vWF is a large plasma glycoprotein which promotes the adhesion of platelets to the vessel wall after a vascular injury. vWF is initially synthesized as a pre-pro-polypeptide. During its transport to the outside of the cell, the single-chain polypeptides are assembled into multimers. The pro-polypeptide can be cleaved and also be secreted. Free pro-polypeptide is identified as von Willebrand antigen II, a plasma glycoprotein of unknown function. Plasma vWF consists of a heterogenous series of multimers, composed of an apparently single-type glycoprotein subunit, linked together by disulfide bonds. The hemostatic potency of vWF was shown to increase with increasing multimer size. Therefore, the multimeric assembly of vWF is a crucial aspect in vWF biosynthesis. Furthermore, vWF synthesized by endothelial cells can either be secreted constitutively or stored and released upon stimulation of the endothelial cell. In this review, data are presented which contribute to the understanding of the biosynthetic pathway and complex processing which vWF has to undergo before it is secreted by the endothelial cell. These data have allowed a prediction of the sequential events underlying vWF biosynthesis, processing, multimer assembly, and secretion.     

Canine von Willebrand factor expresses a multimeric composition similar to human von Willebrand factor

Canine von Willebrand factor (vWf) was compared to human vWf. Antisera raised against human vWf or canine vWf cross-reacted with both heterologous proteins and reactions of partial identity were seen using crossed immunoelectrophoresis. Similar patterns of multimerization were obtained for vWf from both canine and human sources using the enzyme-linked immunoelectrotransfer blot method. However, the canine protein displayed an altered electrophoretic mobility. The molecular weight of the vWf monomer was estimated by SDS-PAGE and found to be indistinguishable from that of human vWf monomer. Canine vWf is decreased in animals with clinically evident hypothyroidism and in heterozygous "carriers" of von Willebrand's disease (vWd) that display no clinical symptoms of vWd. Results expand the concept that von Willebrand's disease in dogs may be a useful model for study of vWd in humans, and that immunochemical methods established for studies of human vWf appear appropriate for studies of canine vWf.     

Hybridoma antibodies to human von Willebrand factor

Twenty-seven stable subclones of seven independent cellular hybrids producing murine monoclonal antibodies to human von Willebrand factor (vWF) have been established. The specificity of the hybridoma antibodies for vWF has been substantiated by a variety of methods including binding to highly purified vWF, absence of binding to plasma or cryoprecipitate from severe von Willebrand's disease, binding to different size multimers in normal plasma, and binding to low molecular weight multimers in type IIA von Willebrand's disease plasma. Monoclonality of the hybridoma derived antibodies has been sought by repeated cloning at limiting dilutions. All seven specificities of hybridoma antibodies cross-react to a variable degree with porcine vWF and all but one with bovine vWF, indicating that they bind to structural loci that are relatively though not identically conserved between species. Hybridoma antibodies bind native as well as denatured vWF, suggesting that the epitopes may be determined to a considerable extent by primary structure rather than entirely by tertiary or quaternary conformation. The hybridoma antibodies define precise epitopes in the vWF molecule, extend the horizon of direct analysis of vWF to the intramolecular level, and may be useful in assigning functional loci to parts of the molecule.     

Immunostaining of von Willebrand factor multimers on agarose gels and nitrocellulose filters

Human von Willebrand factor (VWF) multimeric analysis is commonly performed by agarose gel electrophoresis, electroblotting, and immunoenzymatic staining; however, high molecular weight (HMW) multimers are poorly transferred on nitrocellulose and should be visualized by direct gel staining with radiolabeled anti-VWF antibody and autoradiography or luminography.     

Hybridoma antibodies to human von Willebrand factor

Hybridoma antibodies specific for seven independent topographical sites were used to characterize von Willebrand factor (vWF) and to relate the epitopes to functional loci required for vWF-mediated adhesion of platelets to subendothelium and ristocetin-induced platelet aggregation. The capacity of antibodies to influence the adhesion of human platelets to rabbit aortic subendothelium was analysed in annular perfusion chambers. At a high shear rate similar to that of the microcirculation, four monoclonal antibodies inhibited adhesion. In contrast, no inhibition was observed at low shear. Only one of the four antibodies that inhibited platelet adhesion also attenuated ristocetin-cofactor activity (VIIIR:RCo). Conversely, one antibody that inhibited VIIIR:RCo had no effect upon platelet adhesion. These data support the hypothesis that the molecular loci involved in the two biological functions of vWF are not identical. When these conclusions are considered within the context of a spatial map of the vWF protein surface developed by competitive displacement analysis, the epitopes related to platelet adhesion appear to be spaced and differ from those involved in ristocetin-induced platelet-platelet interaction.     

Study of human von Willebrand factor immunogenicity in pigs with severe von Willebrand disease.

Replacement therapy is the treatment of choice for patients with von Willebrand disease who are unresponsive to desmopressin. In order to prevent transmission of non-enveloped viruses, a solvent/detergent-treated plasma-derived von Willebrand factor available in France since 1989 has been subjected to additional removal/inactivation steps by 35 nm filtration and dry heating for 72 h at 80 degrees C. This preclinical study evaluates the potential immunogenicity of this new product by comparing the antibodies raised in pigs affected with von Willebrand disease after intravenous injection of either a solvent/detergent-treated product or a triple-secured product. Our data showed that there is no difference between the two products in terms of the rate and intensity of the humoral response measured by both binding and neutralizing antibody levels. It was concluded that no antigenic alterations of von Willebrand factor molecules during the nanofiltration and final dry-heating steps were detected in our animal model.     

Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N-terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin- or ristocetin-stimulated platelets, ¹²⁵I-plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5-fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin-stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.     

Optimizing therapy with factor VIII/von Willebrand factor concentrates in von Willebrand disease

In von Willebrand disease, the main goals of treatment are to correct the dual defect of haemostasis caused by a reduced or abnormal von Willebrand factor (vWF), i.e. the prolonged bleeding time (BT) and the deficiency of factor VIII coagulant activity (FVIII:C). The synthetic vasopressin analogue, desmopressin (DDAVP), has reduced the need for transfusions in most of the mild forms of von Willebrand disease but DDAVP is ineffective in type 3 and in other severe cases of types 1 and 2 von Willebrand disease. For many years cryoprecipitate has been the mainstay of replacement therapy but, after the introduction of virucidal methods, concentrates containing FVIII/vWF have been considered much safer than cryoprecipitate and proposed in von Willebrand disease management. FVIII/vWF concentrates have been produced and tested by many authors but there is only one report describing four virus-inactivated FVIII/vWF concentrates evaluated in a cross-over randomized trial. According to these in vitro and pharmacokinetic data, the following information can be derived: (a) no FVIII/vWF concentrate had an intact multimeric structure similar to that of normal plasma or of cryoprecipitate; (b) all FVIII/vWF concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed in the concentrate devoid of FVIII:C; (c) no FVIII/vWF concentrate consistently normalized the BT in a sustained fashion. On the other hand, clinical haemostasis can be achieved in the management of bleeding episodes and of surgery for most of von Willebrand disease cases regardless of whether the BT is corrected; in the few rare cases with mucosal bleeding not controlled by FVIII/vWF concentrates, infusion of DDAVP or platelet concentrates can be administered in addition.     

Zebrafish von Willebrand factor

von Willebrand factor (vWF) is a large protein involved in primary hemostasis. A dysfunction in this protein or an insufficient production of the protein leads to improper platelet adhesion/aggregation, resulting in a bleeding phenotype known as von Willebrand disease (vWD). To gain a better understanding of vWF interactions in vivo, the use of zebrafish as a model is ideal because of the transparency of the embryos and larvae. In this article, we examined the presence and function of vWF in hemostasis of zebrafish utilizing a variety of molecular methods. Using RT-PCR and antibody staining, we have shown that vWF mRNA is present in thrombocytes. Through antibody staining, we demonstrated vWF is synthesized in blood vessels. The role of zebrafish vWF in hemostasis was established through knockdown methods using vWF morpholino (vWF MO) antisense oligonucleotides. Embryos injected with vWF MO at the one to four cell stages resulted in a bleeding phenotype. Injection of embryos with vWF MO also caused an increase in time to occlusion within arteries in larvae upon laser induced injury. We then used vWF-specific Vivo-morpholinos (VMO) to induce vWF knockdown in adult zebrafish by targeting the exon homologous to the human exon 28 of the vWF gene. The reduced ristocetin-mediated agglutination of thrombocytes in a plate tilting assay, using blood from adult zebrafish injected with VMO, provided evidence that vWF is involved in the hemostatic process. We also administered desmopressin acetate to larvae and adults which resulted in enhanced aggregation/agglutination of thrombocytes. Zebrafish genome database analysis revealed the presence of GPIbβ gene. It also revealed the exon of zebrafish vWF gene corresponding to exon 28 of human vWF gene is highly similar to the exon 28 of human vWF gene, except that it has an insertion that leads to a translated peptide sequence that separates the two A domains coded by this exon. This exon is also conserved in other fishes. In summary, we established that zebrafish vWF has a role similar to that of vWF found in humans, thus, making zebrafish a useful model for studying the cell biology of vWF in vivo.     

Increased factor VIII/von Willebrand factor antigen and von Willebrand factor activity in renal failure.

Factor VIII/von Willebrand factor antigen and von Willebrand factor activity (ristocetin assay) were studied in 12 patients in renal failure. A dramatic increase in both activities was observed (antigen 315 +/- 30 per cent in patients verus 104 +/- 9 per cent in control subjects; activity 402 +/- 48 per cent in patients versus 111 +/- 5 per cent in control subjects; p less than 0.001 for both). Since von Willebrand factor is thought to play at least a facilitative role in the development of arteriosclerosis, these increased activities may contribute to the premature arteriosclerosis reported in patients with chronic renal failure undergoing dialysis.     

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL^?1 per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16–60 years), and duration of follow‐up ranged from 5 to 26 years (mean 11 years). Patients had 2–10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU mL^?1 0.34 IU mL^?1 and 0.75 IU mL^?1. At last follow‐up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU L^?1, 0.56 IU mL^?1 and 0.90 IU mL^?1 (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL^?1 (0.21–0.39, CI 95%, P < 0.0001), 0.20 IU mL^?1 per decade (0.13–0.27, CI 95%, P = 0.0001) and 0.20 IU mL^?1 (0.11–0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age‐related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.     

Sulfation of von Willebrand factor

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated [35S]-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate-sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.     

Shear-dependent changes in the three-dimensional structure of human von Willebrand factor

The three-dimensional tertiary structure of human von Willebrand Factor (vWF) on a hydrophobic surface under aqueous conditions and different shear stress regimes was studied by atomic force microscopy (AFM). vWF was imaged by AFM at molecular level resolution under negligible shear stress, under a local applied shear force (7.4 to 19 nN) using the AFM probe in contact mode scanning, and after subjecting vWF to a range of shear stress (0 to 42.4 dyn/cm2) using a rotating disk system. The results demonstrate that vWF undergoes a shear stress-induced conformational transition from a globular state to an extended chain conformation with exposure of intra-molecular globular domains at a critical shear stress of 35 +/- 3.5 dyn/cm2. The globular vWF conformation (149 nm by 77 nm and height 3.8 nm) is representative of native vWF after simple diffusion to the hydrophobic surface, followed by adhesion and some spreading. In a shear stress field above the critical value, protein unfolding occurs and vWF is observed in extended chain conformations oriented in the direction of the shear stress field with molecular lengths ranging from 146 to 774 nm and 3.4 nm mean height. The shear stress-induced structural changes to vWF suggest a close conformation-function relationship in vWF properties for thrombogenesis in regions of high shear stress.     

Reduced von Willebrand factor survival in type Vicenza von Willebrand disease.

Type Vicenza variant of von Willebrand disease (VWD) is characterized by a low plasma von Willebrand factor (VWF) level and supranormal VWF multimers. Two candidate mutations, G2470A and G3864A at exons 17 and 27, respectively, of the VWF gene were recently reported to be present in this disorder. Four additional families, originating from northeast Italy, with both mutations of type Vicenza VWD are now described. Like the original type Vicenza subjects, they showed a mild bleeding tendency and a significant decrease in plasma VWF antigen level and ristocetin cofactor activity but normal platelet VWF content. Unlike the original patients, ristocetin-induced platelet aggregation was found to be normal. Larger than normal VWF multimers were also demonstrated in the plasma. Desmopressin (DDAVP) administration increased factor VIII (FVIII) and VWF plasma levels, with the appearance of even larger multimers. However, these forms, and all VWF oligomers, disappeared rapidly from the circulation. The half-life of VWF antigen release and of elimination was significantly shorter than that in healthy counterparts, so that at 4 hours after DDAVP administration, VWF antigen levels were close to baseline. Similar behavior was demonstrated by VWF ristocetin cofactor activity and FVIII. According to these findings, it is presumed that the low plasma VWF levels of type Vicenza VWD are mainly attributed to reduced survival of the VWF molecule, which, on the other hand, is normally synthesized. In addition, because normal VWF-platelet GPIb interaction was observed before or after DDAVP administration, it is proposed that type Vicenza VWD not be considered a 2M subtype.     

Immunogold labelling of human von Willebrand factor adsorbed to collagen

von Willebrand factor (vWF) mediates adhesion of platelets to the exposed subendothelium at sites of vascular injury. This function is expressed through binding of vWF to both collagen and receptors on the platelet membrane. We have developed a new method using immunogold staining and electron microscopy, permitting visualization of human vWF adsorbed to collagen fibrils. The electron micrographs revealed strings of gold beads reflecting the polymeric structure of vWF. Our data showed dramatic differences in the binding of vWF to collagens of different sources: high binding density was observed using a collagen preparation isolated from aortic tissue whereas colloidal gold was virtually absent from tendon collagen. Using the immunogold labelling method we demonstrated that high shear rate enhanced vWF binding to aortic collagen.