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.     

血管性血友病因子裂解蛋白酶的稳定表达及其活性检测

本研究旨在得到重组的血管性血友病因子裂解蛋白酶(ADAMTS13),进一步研究其在血栓止血中的作用。利用脂质体将编码ADAMTS13全长序列的重组质粒pSecTag-ADAMTS13转染Hela细胞,用潮霉素(Hygromycin-B)筛选得到阳性克隆细胞株,并扩大培养,收集上清。利用Ni-NTA琼脂糖柱、梯度咪唑淋洗法纯化蛋白,SDS-PAGE和Westernblotting鉴定纯化产品纯度和免疫学活性,采用GST-His双抗夹心法测定蛋白剪切活性。结果显示,成功获得一株能恒定分泌重组ADAMTS13蛋白的细胞株ADAMTS2-4,每1L培养上清可纯化得到5.8mg重组蛋白。Western blotting结果显示,ADAMTS13多抗能与重组蛋白在190kDa处显单一条带,并且蛋白具有6.4U/mL的剪切活性(每毫升正常人混合血浆中ADAMTS13活性为1U)。重组蛋白具有较好的免疫原活性和酶活性,为进一步研究ADAMTS13作用机理和运用奠定了良好的基础。     

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.     

The propeptide of von Willebrand factor independently mediates the assembly of von Willebrand multimers

The biosynthesis of von Willebrand Factor (vWF) by vascular endothelial cells involves a complex series of processing steps that includes proteolytic cleavage of a 741-residue propeptide and the assembly of disulfide-linked multimers. Using a model system in which experimentally altered vWF cDNAs are expressed in COS-1 cells, we have shown that the vWF propeptide contains determinants that govern the assembly of vWF multimers. Furthermore, the role of the propeptide (in the assembly process) does not require it to be a contiguous part of the pro-vWF primary structure, since independently expressed propeptide was shown to promote the assembly of mature vWF subunits into multimers. Pulse-chase experiments indicated that the independently expressed propeptide formed a transient association with the mature vWF subunit inside the cell. Thus, it appears that the vWF propeptide segment can act in "trans" to direct the assembly of disulfide-linked vWF multimers.     

Intracellular storage and regulated secretion of von Willebrand factor in quantitative von Willebrand disease

Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.     

Ristocetin-induced self-aggregation of von Willebrand factor

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.     

Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues.

Few systematic studies have been published comparing the expression and distribution of endothelial cell (EC) markers in different vascular beds in normal human tissues. We investigated by immunohistochemistry the expression of CD31, CD34, von Willebrand factor (vWF), and Fli-1 in EC of the major organs and large vessels. Tissue samples obtained from autopsies and biopsy specimens were routinely processed and stained immunohistochemically for CD31, CD34, and vWF. Biopsy material was also stained immunohistochemically for Fli-1, D2-40, and Lyve-1. The expression pattern of the markers was heterogeneous in some of the organs studied. In the kidney, fenestrated endothelium of the glomeruli strongly expressed CD31 and CD34 but was only focally positive or completely negative for vWF. Alveolar wall capillaries of the lung strongly stained for CD31 and CD34 but were usually negative for vWF. The staining intensity for vWF increased gradually with the vessel caliber in the lung. Sinusoids of the spleen and liver were diffusely positive for CD31. They were negative for CD34 in the spleen and only expressed CD34 in the periportal area in the liver. Fli-1 was expressed in all types of EC but also in lymphocytes. D2-40 stained lymphatic endothelium only. Lyve-1 immunostaining was too variable to be applied to routinely processed tissues. The expression of EC markers CD31, CD34, and vWF in the vascular tree is heterogeneous with a specific pattern for individual vessel types and different anatomic compartments of the same organ. D2-40 labels lymphatic EC only.     

Association of the factor VIII light chain with von Willebrand factor

Coagulation factor VIII (fVIII) is isolated from porcine blood as a set of three heterodimers because of proteolytic cleavages in the middle, or B region, of the parent single-chain molecule. A single 80-kDa COOH-terminal fragment, the light chain (fVIIILC), is associated with one of three forms of heavy chain (fVIIIHCs) by a calcium-dependent linkage. The purified heterodimers were dissociated using EDTA and fVIIILC, and fVIIIHCs were isolated by high pressure liquid chromatography under nondenaturing conditions. The association of fVIII, fVIIILC, and fVIIIHCs with multimeric human von Willebrand factor (vWF) was studied using analytical velocity sedimentation. A previous study using this method with an intact, single heterodimeric species of fVIII has shown that one molecule of fVIII can bind to each subunit of vWF (Lollar, P., and Parker, C.G. (1987) J. Biol. Chem. 262, 17572-17576). fVIIILC bound vWF as judged by the increase in the plateau height and sedimentation coefficient of the fVIIILC.vWF complex compared to vWF at 42,000 x g and by the decrease in the plateau height of the 4.8 S fVIIILC boundary sedimenting at 240,000 x g. Titration of a fixed concentration of fVIIILC with vWF yielded a stoichiometry of one fVIIILC molecule per subunit of vWF. Proteolytic cleavage by thrombin to remove an acidic 41-residue NH2-terminal peptide from fVIIILC completely abolished its binding to vWF. In contrast, no binding of fVIIIHCs to vWF was observed. Additionally, intact fVIII bound to vWF was completely dissociated after proteolysis by thrombin. These data are consistent with the hypothesis that a critical step in blood coagulation is the release of all regions of fVIII from vWF following a single proteolytic cleavage of fVIIILC.     

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.     

Crystal structure of the von Willebrand factor modulator botrocetin

The binding of von Willebrand factor (vWF) to the platelet receptor, glycoprotein (GP) Ib-IX-V complex, has a key role in the initiation of thrombus formation and is regulated by interactions with extracellular matrix components under the influence of hemodynamic forces. To a certain extent, these effects can be mimicked in vitro by two nonphysiologic modulators, ristocetin and botrocetin. The latter, isolated from the venom of the snake Bothrops jararaca, is a 31-kDa heterodimeric protein that forms a soluble complex with vWF. As an initial step toward understanding the mechanisms that regulate vWF function, we have solved the crystal structure of botrocetin at 1.8 A resolution. Botrocetin exhibits homology with other snake proteins, but contains only one metal binding site as compared to two in Factor IX binding protein and Factor IX/X binding protein and none in flavocetin. A distinctive feature of botrocetin is the presence of a negatively charged surface that may play a role in the association with the vWF A1 domain.     

Thermodynamic analysis of the interaction of factor VIII with von Willebrand factor

Factor VIII (FVIII) is a glycoprotein that plays an important role in the intrinsic pathway of coagulation. In circulation, FVIII is protected upon binding to von Willebrand factor (VWF), a chaperone molecule that regulates its half-life, distribution, and activity. Despite the biological significance of this interaction, its molecular mechanisms are not fully characterized. We determined the equilibrium and activation thermodynamics of the interaction between FVIII and VWF. The equilibrium affinity determined by surface plasmon resonance was temperature-dependent with a value of 0.8 nM at 35 °C. The FVIII-VWF interaction was characterized by very fast association (8.56 × 10(6) M(-1) s(-1)) and fast dissociation (6.89 × 10(-3) s(-1)) rates. Both the equilibrium association and association rate constants, but not the dissociation rate constant, were dependent on temperature. Binding of FVIII to VWF was characterized by favorable changes in the equilibrium and activation entropy (TΔS° = 89.4 kJ/mol, and -TΔS(++) = -8.9 kJ/mol) and unfavorable changes in the equilibrium and activation enthalpy (ΔH° = 39.1 kJ/mol, and ΔH(++) = 44.1 kJ/mol), yielding a negative change in the equilibrium Gibbs energy. Binding of FVIII to VWF in solid-phase assays demonstrated a high sensitivity to acidic pH and a sensitivity to ionic strength. Our data indicate that the interaction between FVIII and VWF is mediated mainly by electrostatic forces, and that it is not accompanied by entropic constraints, suggesting the absence of conformational adaptation but the presence of rigid "pre-optimized" binding surfaces.     

Amino acid sequence of human von Willebrand factor

The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.     

Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.     

Subunit composition of oligomeric human von Willebrand factor

The oligomerization of human endothelial cell-synthesized von Willebrand factor (vWf) has been studied by gel chromatography in columns of Sephacryl S-500 and by discontinuous agarose gel electrophoresis. A quantitative recovery of high Mr vWf oligomers has been obtained after binding to a monoclonal anti-vWf-Sepharose adduct. This reagent has been used to analyze gel filtration chromatographic elution profiles of [35S]methionine-labeled culture medium and cell lysate. It was determined that high Mr oligomers are present in endothelial cell lysates as well as in the medium overlying these cells and are composed of Mr 225,000 subunits. When vWf oligomers were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of a reducing agent, the Mr 240,000 subunit (provWf) was not observed to oligomerize beyond the dimer stage to a significant degree. Therefore, vWf oligomerization appears to be facilitated by conversion of provWf subunits to mature vWf subunits, most likely by proteolytic removal of sequences unique to the intracellular precursor.