Effects of the mutant von Willebrand factor gene in von Willebrand disease

Von Willebrand disease (vWD) is a common inherited bleeding disorder in humans, and can be divided into a mild (type 1) and severe (type 3) form. Previous linkage studies identified one subject with vWD type 1 who transmitted different alleles of the von Willebrand factor (vWF) gene to his two affected children, one having vWD type 3 and the other having type 1. By screening the promoter and coding sequence (52 exons) of the vWF gene, three missense mutations were detected in this family. The type 1 individual who transmitted different alleles of the gene to his two sick children carries two substitutions, one in exon 5 and the other in exon 18 on the respective alleles. The relationship between the genotype (mutations) and the phenotype in this family is complex. In order further to correlate the relationship in vWD type 1 individuals, fifty-five subjects who carry one null allele of the vWF gene were collected. All these subjects are from vWD type 3 families with known mutations. Biochemical data of these 55 subjects indicate that gene dosage and other factors, such as blood group, age, and environment factors, play a critical role in the development of the phenotype of the disease.     

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.     

Type-3 von willebrand's disease in a rhesus monkey (Macaca mulatta)

Severe type-3 von Willebrand's disease (vWD) was diagnosed in a young male rhesus monkey that had excessive bleeding from minor wounds. Plasma samples from the monkey had no detectable quantitative or functional von Willebrand factor (vWF), low Factor-VIII coagulant activity, and moderate prolongation of activated partial thromboplastin time. Testing of the affected monkey's extended family revealed a likely hereditary basis for the vWD, in that the sire and a paternal half-sister had markedly reduced plasma vWF concentration. Fresh whole blood was transfused to control frequent bleeding episodes throughout the monkey's life. Although vWD is the most common inherited bleeding disorder in humans and dogs, this is the first report of vWD in a nonhuman primate.     

Characterization of recombinant von Willebrand factor corresponding to mutations in type IIA and type IIB von Willebrand disease.

Type IIA and IIB von Willebrand disease (vWD) result from defects in von Willebrand factor (vWF). Although both type IIA and IIB vWD are characterized by the absence of high molecular weight multimers in plasma, vWF from patients with type IIA vWD demonstrates a decreased affinity for the platelet receptor glycoprotein Ib (GPIb), whereas vWF from patients with type IIB vWD show an increased affinity for GPIb. To investigate how structural alterations in vWF affect its interaction with GPIb, we reproduced the reported potential mutations in type IIA and IIB vWD in vWF cDNA and expressed the recombinant proteins in COS-1 cells. The type IIA vWF potential mutation was represented by a G-->A transversion which results in the substitution of Lys for Glu at position 875 in the mature vWF subunit (rvWFLys875). The type IIB vWF mutation corresponds to a duplicated ATG codon, resulting in three contiguous methionines starting at position 540-541 in the normal vWF sequence (rv-WFduplMet540-541). The subunit composition and multimeric structure of both mutant proteins were similar to the wild-type rvWF. The rvWFLys875 bound to fixed platelets in the presence of ristocetin similar to wild-type rvWF. The rvWFduplMet540-541 bound to fixed platelets in the absence of agonist. The rvWFLys875 appears to interact normally with GPIb, and the decreased affinity for the platelet receptor observed in plasma is probably a consequence of prior reduction in multimeric size resulting from the defect. In contrast, the duplication of Met540-541 increases the reactivity of vWF for its platelet receptor.     

Impaired intracellular transport produced by a subset of type IIA von Willebrand disease mutations.

Type IIA von Willebrand disease (vWD) results from abnormalities in von Willebrand factor (vWF) characterized by absence of plasma high molecular weight (HMW) vWF multimers. In this report, 5 distinct point mutations were identified in 6 Type IIA vWD families. A total of 7 mutations, all clustered within a 124-amino acid segment of the vWF A2 domain, now account for 9 of a panel of 11 Type IIA families. In COS-7 cells, 3 single amino acid substitutions, Val844----Asp, Ser743----Leu, and Gly742----Arg, impaired the transport of vWF multimers between the endoplasmic reticulum and the Golgi complex, with more profound effects on the secretion of HMW multimers than lower molecular weight forms. In contrast, 2 substitutions, Arg834----Trp and Gly742----Glu, resulted in secretion of HMW multimers similar to wild-type vWF. The vWF structure observed within patient platelets correlated closely with the synthesis pattern seen for the corresponding mutants in COS-7 cells. These findings demonstrate that structural alterations within the A2 domain of vWF can produce the characteristic phenotype of Type IIA vWD via two distinct molecular mechanisms.     

Genetic and blood coagulation characterization of “Swedish” families with von Willebrand's disease types I and III: New aspects of heredity

Summary Twenty-five patients with von Willebrand's disease (vWD) type III were analysed with regard to blood coagulation variables and possible deletions. Nine of the probands and their families were further investigated with DNA linkage analyses. Different patterns of heredity can be suggested in our families with vWD type III, on the basis of blood coagulation analyses. The findings suggest homozygosity in five families and the possibility of compound heterozygosity or a new mutation in the proband in three families. The linkage analyses confirm the results of the coagulation analyses. The segregation of the von Willebrand factor (vWF) gene can be followed in the families, and carrier diagnosis can be made in several of the probands' relatives. The possibility of large deletions in the vWF gene of the probands and their parents was investigated with probes representing the whole vWF cDNA. No deletions were found.This study was approved by the Ethics Committee of the Karolinska Hospital (No. 84:1)     

Molecular characterization of a unique von Willebrand disease variant. A novel mutation affecting von Willebrand factor/factor VIII interaction

von Willebrand factor (vWF) plays a central role in blood coagulation, mediating the adhesion of the initial platelet plug to the subendothelium, and serving as the carrier for factor VIII (FVIII) in the circulation. In previous studies, we have mapped the epitope for an anti-vWF monoclonal antibody which inhibits the interaction between FVIII and vWF to a region spanning Thr78 to Thr96 of the mature protein (Bahou, W.F., Ginsburg, D., Sikkink, R., Litwiller, R., and Fass, D. N. (1989) J. Clin. Invest. 84, 56-61). We now report the identification of a mutation within this region of vWF that results in decreased FVIII binding. Sequence analysis of polymerase chain reaction amplified platelet vWF mRNA from a von Willebrand disease (vWD) patient with a disproportionately low FVIII level identified a single nucleotide substitution (G----A), resulting in the conversion of Arg91----Gln. Recombinant vWF carrying this substitution showed decreased binding to FVIII compared with wild-type vWF or vWF carrying a polymorphic substitution in the same region (Arg89----Gln). These observations suggest a critical role for Arg91 in the interaction of vWF with FVIII and identify the molecular mechanism for a variant of vWD associated with unusually low FVIII levels.     

内含肽介导三段vWF基因真核细胞转移的翻译后连接及其功能性多聚体形成

vWF(von Willebrand factor)是一种超大分子质量的血浆多聚体糖蛋白,在血栓形成和生理凝血过程中发挥重要作用,其质和/或量的缺陷导致血管性血友病(VWD),由于VWD为单基因病,且vWF为分泌性蛋白,基于基因转移的基因治疗无需特异的靶器官,因此VWD特别适合于基因治疗,但vWF基因过大(8.4 kb),难以为多数病毒载体特别是优点较多的腺相关病毒(AAV)载体承载.运用内含肽(intein)的蛋白质反式剪接功能,研究了三重载体真核细胞共转断裂3段的vWF基因,以期通过转基因翻译后的蛋白质剪接作用形成完整的功能性vWF蛋白.将vWF的cDNA于满足剪接所需的保守性氨基酸Cys1099、Ser2004的密码子前断裂为3段,分别与2种不同的内含肽即Ssp DnaE内含肽和Ssp DnaB内含肽编码序列融合,构建到真核表达载体pcDNA3.1(+),得到3个分别融合内含肽的vWF片段基因真核表达载体,共转染培养的293细胞,通过瞬时表达,电泳观察培养上清中的vWF多聚体形态,分析vWF蛋白量和凝血Ⅷ因子(FⅧ)结合力;通过共转FⅧ基因,分析了培养上清中的FⅧ蛋白量及生物活性.结果显示,通过内含肽的蛋白质反式剪接作用,共转内含肽融合的三片段vWF基因细胞上清,表现与正常人血浆和转vWF基因阳性对照细胞相似的vWF多聚体模式和FⅧ结合力,而且可明显提高转FⅧ基因后表达的FⅧ蛋白的分泌量和活性,提示剪接vWF蛋白的FⅧ载体功能的恢复.结果表明,内含肽可作为一种有效的技术手段进行三重载体共转断裂的vWF基因,为进一步基于内含肽的三重AAV转断裂vWF基因应用于VWD基因治疗研究、克服AAV的容量限制提供了依据.     

Acquired von Willebrand's disease in the course of severe primary hypothyroidism in a patient with autoimmune polyglandular syndrome type 3

The case of a 20-year old female, who had been followed because of von Willebrand disease (vWD) was presented in this paper . She had a past history of menorrhagia and bleeding after dental procedures and the activity of von Willebrand factor (vWF) was decreased. Because of suggestive clinical features, the workup for hypothyroidism was performed and the patient was found to have severe hypothyroidism due to Hashimoto thyroiditis. After the institution of replacement therapy with levothyroxine, von Willebrand factor activity returned to normal range and symptoms of von Willebrand disease disappeared. Based on these findings, the diagnosis of acquired von Willebrand syndrome (AvWS) due to hypothyroidism was made. The development of myasthenia led to the final diagnosis of autoimmune polyglandular syndrome type 3 (APS) with myasthenia gravis and vitiligo.     

Misfolding of vWF to Pathologically Disordered Conformations Impacts the Severity of von Willebrand Disease

The primary hemostatic von Willebrand factor (vWF) functions to sequester platelets from rheological blood flow and mediates their adhesion to damaged subendothelium at sites of vascular injury. We have surveyed the effect of 16 disease-causing mutations identified in patients diagnosed with the bleeding diathesis disorder, von Willebrand disease (vWD), on the structure and rheology of vWF A1 domain adhesiveness to the platelet GPIbα receptor. These mutations have a dynamic phenotypical range of bleeding from lack of platelet adhesion to severe thrombocytopenia. Using new rheological tools in combination with classical thermodynamic, biophysical, and spectroscopic metrics, we establish a high propensity of the A1 domain to misfold to pathological molten globule conformations that differentially alter the strength of platelet adhesion under shear flow. Rheodynamic analysis establishes a quantitative rank order between shear-rate-dependent platelet-translocation pause times that linearly correlate with clinically reported measures of patient platelet counts and the severity of thrombocytopenia. These results suggest that specific secondary structure elements remaining in these pathological conformations of the A1 domain regulate GPIbα binding and the strength of vWF-platelet interactions, which affects the vWD functional phenotype and the severity of thrombocytopenia.     

Recombinant von Willebrand factor Arg578-->Gln. A type IIB von Willebrand disease mutation affects binding to glycoprotein Ib but not to collagen or heparin.

von Willebrand factor (vWF) is a multimeric plasma glycoprotein that mediates platelet adhesion to the subendothelium via binding to platelet glycoprotein Ib (GPIb) and to components of the vessel wall. Recently, missense mutations that cause type IIB von Willebrand disease (vWD) were described, clustered within a disulfide loop in the A1 domain of vWF that has binding sites for GPIb, collagen, and heparin. In type IIB vWD, plasma vWF exhibits increased affinity for platelet GPIb, but decreased binding to collagen and heparin. The effect was studied of a type IIB vWD mutation, Arg578-->Gln, on the interaction of vWF with GPIb, collagen, and heparin. Recombinant wild type rvWF and mutant rvWF(R578Q) were expressed in COS-7 cells. Ristocetin-induced binding of rvWF(R578Q) to GPIb was markedly increased compared with rvWF, confirming that the Arg578-->Gln mutation causes the characteristic gain-of-function abnormality of type IIB vWD; botrocetin-induced binding was only slightly increased. Binding to collagen type III and heparin-agarose was compared for rvWF(R578Q) and plasma vWF from patients with four different type IIB mutations: Arg543-->Trp, Arg545-->Cys, Val553-->Met, Arg578-->Gln. For all of the plasma samples, binding to collagen and to heparin was reduced compared with normal plasma. In contrast, binding of rvWF(R578Q) to collagen and heparin was normal compared with wild type rvWF. Therefore, the Arg578-->Gln mutation increases the affinity of vWF for GPIb but does not directly impair vWF interaction with collagen or heparin. Arg578 may therefore be necessary to prevent normal vWF from interacting with GPIb. In type IIB vWD, the defective binding of plasma vWF to collagen and heparin may be secondary to post-synthetic modifications that occur in vivo, such as the loss of high molecular weight vWF multimers.     

Misfolding of vWF to Pathologically Disordered Conformations Impacts the Severity of von Willebrand Disease

The primary hemostatic von Willebrand factor (vWF) functions to sequester platelets from rheological blood flow and mediates their adhesion to damaged subendothelium at sites of vascular injury. We have surveyed the effect of 16 disease-causing mutations identified in patients diagnosed with the bleeding diathesis disorder, von Willebrand disease (vWD), on the structure and rheology of vWF A1 domain adhesiveness to the platelet GPIbα receptor. These mutations have a dynamic phenotypical range of bleeding from lack of platelet adhesion to severe thrombocytopenia. Using new rheological tools in combination with classical thermodynamic, biophysical, and spectroscopic metrics, we establish a high propensity of the A1 domain to misfold to pathological molten globule conformations that differentially alter the strength of platelet adhesion under shear flow. Rheodynamic analysis establishes a quantitative rank order between shear-rate-dependent platelet-translocation pause times that linearly correlate with clinically reported measures of patient platelet counts and the severity of thrombocytopenia. These results suggest that specific secondary structure elements remaining in these pathological conformations of the A1 domain regulate GPIbα binding and the strength of vWF-platelet interactions, which affects the vWD functional phenotype and the severity of thrombocytopenia.     

Polymerase chain reaction amplification of two polymorphic simple repeat sequences within the von Willebrand factor gene: application to family studies in von Willebrand disease

Summary We have used the polymerase chain reaction to amplify two variable number of tandem repeats (VNTRs) within a region of repetitive DNA located in intron 40 of the von Willebrand factor (vWf) gene. Heterozygosity for VNTR I was observed in 30 out of 39 normal unrelated individuals tested (77%), and for VNTR II in 29 out of 44 (66%) similar individuals. Family studies were carried out on 11 kindreds with von Willebrand disease (vWD). Ten of these families were found to be informative for one or other of the VNTRs or for a combination of data from both VNTRs. This method can be used for antenatal diagnosis and for carrier diagnosis in recessive forms of vWD. It is also useful for tracking the gene associated with vWD in type I families where there may be one or more individuals with a phenotypically uncertain diagnosis.     

von Willebrand factor unfolding mediates platelet deposition in a model of high-shear thrombosis

Thrombosis under high-shear conditions is mediated by the mechanosensitive blood glycoprotein von Willebrand factor (vWF). vWF unfolds in response to strong flow gradients and facilitates rapid recruitment of platelets in flowing blood. While the thrombogenic effect of vWF is well recognized, its conformational response in complex flows has largely been omitted from numerical models of thrombosis. We recently presented a continuum model for the unfolding of vWF, where we represented vWF transport and its flow-induced conformational change using convection-diffusion-reaction equations. Here, we incorporate the vWF component into our multi-constituent model of thrombosis, where the local concentration of stretched vWF amplifies the deposition rate of free-flowing platelets and reduces the shear cleaning of deposited platelets. We validate the model using three benchmarks: in vitro model of atherothrombosis, a stagnation point flow, and the PFA-100, a clinical blood test commonly used for screening for von Willebrand disease (vWD). The simulations reproduced the key aspects of vWF-mediated thrombosis observed in these experiments, such as the thrombus location, thrombus growth dynamics, and the effect of blocking platelet-vWF interactions. The PFA-100 simulations closely matched the reported occlusion times for normal blood and several hemostatic deficiencies, namely, thrombocytopenia, vWD type 1, and vWD type 3. Overall, this multi-constituent model of thrombosis enables macro-scale 3D simulations of thrombus formation in complex geometries over a wide range of shear rates and accounts for qualitative and quantitative hemostatic deficiencies in patient blood.     

Germ-line mosaicism for a valine-to-methionine substitution at residue 553 in the glycoprotein Ib-binding domain of von Willebrand factor, causing type IIB von Willebrand disease.

The origin of new single-gene mutations resulting in inherited disease is an issue which may be at least partially resolved by our enhanced ability to detect these changes. In this report we describe the identification of a missense mutation at codon 553 (guanine to adenine) in the von Willebrand factor (vWf) gene in affected members of a family with type IIB von Willebrand's disease (vWd). We found no evidence for this substitution in 190 normal vWf genes. The encoded substitution of a methionine for a valine at this residue is nonconservative in nature and has affected a vWf protein region which has been shown to facilitate binding to the platelet receptor glycoprotein Ib. In patients with type IIB vWd this interaction is characteristically increased in affinity. This mutation has also recently been recorded in four other type IIB vWd families. Thus, there is strong circumstantial evidence to incriminate this substitution as the disease causing mutation in this family. As further supporting evidence for this claim, we have shown by vWf polymorphism analysis that the mutation originated in a vWf gene transmitted from a phenotypically normal grandfather. Analysis of the sperm from this individual showed that approximately 5% of the germ line contained the mutant 553 sequence. These results confirm (1) that the candidate type IIB vWd mutation in this family occurred at some time during the development of the germ line of the grandfather and presumably was related to a mitotic cell division and (2) that, as a result, he is a low-level germ-line mosaic for the mutation.     

Biochemical and molecular characterization of von Willebrand disease type 2N in a pregnant patient who gave birth under analgesia with remifentanil

von Willebrand 's disease (vWD) is the commonest inherited bleeding disorder. Although in literature there are some cases reported of epidural analgesia for labor pain in pregnancies with Von Willebrand's disease, the technique is not free from risk of neurolocal complications. Authors reported a case of spontaneous labor in a pregnant woman with type II vWD, delivered under local analgesia administered through a continuous intravenous infusion of remifentanil integrated by boli. A 34-year-old woman at the 39th week of her second pregnancy was admitted for an active labor of a single fetus in cephalic presentation. The patient had been diagnosed with type II vWD by a hematologist during her first pregnancy. The patient coagulation panel was as follows: a reduction of VIIIth factor concentration (21 percent); a normal value of vWD functional assay; an increase of vWf:Ag (antigen) and a reduction of XIth factor. During labor she was put on remifentanil in PCA (patient controlled analgesia), administered with slow boli followed by continuous infusions at increasing doses. The woman delivered a female fetus weighing 3,550 g, in vertex presentation, in left anterior occipital position, with an A.P.G.A.R. of 8 at the first minute and 9 at the fifth minute. The total duration of labor was 3 hours and 10 minutes. The patient was satisfied with analgesia in labor. The bleeding during and after delivery was regular. In the authors ' opinion, it is important to know that an alternative to epidural analgesia can be used in order to avoid the risk of neurological complications in labor pain for patients with type II Von Willebrand's disease.     

Von Willebrand factor and von Willebrand disease

von Willebrand disease (vWD) is caused by quantitative and/or qualitative defects of the von Willebrand factor (vWF), a multimeric high molecular weight glycoprotein. Typically, it affects the primary hemostatic system, which results in a mucocutaneous bleeding tendency simulating a platelet function defect. The vWF promotes its function in two ways: (i) by initiating platelet adhesion to the injured vessel wall under conditions of high shear forces, and (ii) by its carrier function for factor VIII in plasma. Accumulating knowledge of the different clinical phenotypes and the pathophysiological basis of the disease translated into a classification that differentiated between quantitative and qualitative defects by means of quantitative and functional parameters, and by analyzing the electrophoretic pattern of vWF multimers. The advent of molecular techniques provided the opportunity for conducting genotype-phenotype studies which have recently helped, not only to elucidate or confirm important functions of vWF and its steps in post-translational processing, but also many disease causing defects. Acquired von Willebrand syndrome (avWS) has gained more attention during the recent years. An international registry was published and recommendation by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis in 2000. It concluded that avWS, although not a frequent disease, is nevertheless probably underdiagnosed. This should be addressed in future prospective studies. The aim of treatment is the correction of the impaired hemostatic system of the patient, ideally including the defects of both primary and secondary hemostasis. Desmopressin is the treatment of choice in about 70% of patients, mostly with type 1, while the others merit treatment with concentrates containing vWF.     

Conformational energy analysis of the substitution of Val for Gly 233 in a functional region of platelet GPIb alpha in platelet-type von Willebrand disease

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder in which patient platelets exhibit an abnormally increased binding of circulating von Willebrand factor (vWF). We have recently shown that this abnormality is associated with a point mutation resulting in substitution of Val for Gly 233 in platelet membrane glycoprotein Ib alpha (GPIb alpha), a major component of the platelet GPIb/IX receptor for vWF. To investigate the effect of this substitution on the three-dimensional structure of this region of the protein, we have generated the allowed (low energy) conformations of the region of the GPI alpha protein containing residues 228-238 (with 5 residues on either side of the critical residue 233) with Gly 233 (wild type) and Val 233 (PT-vWD) using the computer program ECEPP (Empirical Conformational Energies of Peptides Program). The wild-type sequence is Tyr-Val-Trp-Lys-Gln-Gly-Val-Asp-Val-Lys-Ala. We find that the Gly 233-containing peptide can exist in two low energy conformers. The lowest energy conformer is a structure containing a beta-turn at Gln 232-Gly 233 while the alternative conformation is an amphipathic helical structure. Only the amphipathic helical structure is allowed for the Val 233-containing peptide which contains a hydrophobic 'face' consisting of Val 229, Val 233 and Val 236 and another hydrophilic surface composed of such residues as Lys 231 and Asp 235. No such surfaces exist for the lowest energy bend conformer for the Gly 233-containing peptide, but do exist in the higher energy helical structure. The amphipathic surfaces in the 228-238 region of the Val 233-containing GPIb alpha protein may associate strongly with complementary surfaces during vWF binding to the GPIb/IX receptor complex and may help explain heightened association of vWF with this receptor in PT-vWD.     

Structure of the gene for human von Willebrand factor

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.