Changing insights in the diagnosis and classification of autosomal recessive and dominant von Willebrand diseases 1980-2015

The European Clinical Laboratory and Molecular (ECLM) criteria define 10 distinct Willebrand diseases (VWD): recessive type 3, severe 1, 2C and 2N; dominant VWD type 1 secretion/clearance defect, 2A, 2B, 2E, 2M and 2D; and mild type 1 VWD (usually carriers of recessive VWD). Recessive severe 1 and 2C VWD are characterized by secretion and multimerization defects caused by mutations in the D1-D2 domain. Recessive 2N VWD is a mild hemophilia due to D’-FVIII-von Willebrand factor (VWF) binding site mutations. Dominant 2E VWD caused by heterozygous missense mutations in the D3 domain is featured by a secretion-clearance-multimerization VWF defect. Dominant VWD type 2M due to loss of function mutations in the A1 domain is characterized by decreased ristocetin-induced platelet aggregation and VWF:RCo, normal VWF multimers and VWF:CB, a poor response of VWF:RCo and good response of VWF:CB to desmopressin (DDAVP). Dominant VWD type 2A induced by heterozygous mutations in the A2 domain results in hypersensitivity of VWF for proteolysis by ADAMTS13 into VWF degradation products, resulting in loss of large VWF multimers with triplet structure of each individual VWF band. Dominant VWD type 2B due to a gain of function mutation in the A1 domain is featured by spontaneous interaction between platelet glycoprotein Ib (GPIb) and mutated VWF A1 followed by increased proteolysis with loss of large VWF multimers and presence of each VWF band. A new category of dominant VWD type 1 secretion or clearance defect due to mutations in the D3 domain or D4-C1-C5 domains consists of two groups: Those with normal or smeary pattern of VWF multimers.     

L1503R is a member of group I mutation and has dominant-negative effect on secretion of full-length VWF multimers: an analysis of two patients with type 2A von Willebrand disease

Summary.  Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF); this in turn is associated with an absence of high-molecular-weight multimers. Sequence analysis of the VWF gene from two unrelated type 2A VWD patients showed an identical, novel, heterozygous T→G transversion at nucleotide 4508, resulting in the substitution of L1503R in the VWF A2 domain. This substitution, which was not found in 60 unrelated normal individuals, was introduced into a full-length VWF cDNA and subsequently expressed in 293T cells. Only trace amount of the mutant VWF protein was secreted but most of the same was retained in 293T cells. Co-transfection experiment of both wild-type and mutant plasmids indicated the dominant-negative mechanism of disease development; as more of mutant DNA was transfected, VWF secretion was impaired in the media, whereas more of VWF was stored in the cell lysates. Molecular dynamic simulations of structural changes induced by L1503R indicated that the mean value of all-atom root-mean-squared-deviation was shifted from those with wild type or another mutation L1503Q that has been reported to be a group II mutation, which is susceptible to ADAMTS13 proteolysis. Protein instability of L1503R may be responsible for its intracellular retention and perhaps the larger VWF multimers, containing more mutant VWF subunits, are likely to be mal-processed and retained within the cell.     

Molecular Genetics of Type 2 von Willebrand Disease

Type 2 von Willebrand disease (VWD) is characterized by a wide heterogeneity of functional and structural defects. These abnormalities' cause either defective von Willebrand factor (VWF)-dependent platelet function in subtypes 2A, 2B, and 2M or defective VWF-factor VIII (FVIII) binding in subtype 2N. The diagnoses of types 2A, 2B, and 2M VWD may be guided by the observation of disproportionately low levels of ristocetin cofactor activity or collagen-binding capacity relative to VWF antigen. The abnormal platelet-dependent function is often associated with the absence of high molecular weight (HMW) multimers (type 2A, type 2B), but the HMW multimers may also be present (type 2M, some type 2B), and supranormal multimers may exist ("Vicenza" variant). The observation of a low FVIII-to-VWF:Ag ratio is a hallmark of type 2N VWD. in which the FVIII levels depend on the severity of the FVIII-binding defect. Today, the identification of mutations in particular domains of the pre-pro-VWF is helpful in classifying these variants and providing further insight into the structure-function relationship and the biosynthesis of VWF. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC VWD. Mutations located in the D' domain or in the N terminus of the D3 domain define type 2N VWD. Mutations in the D3 domain characterize Vicenza and IIE patients. Mutations in the A1 domain may modify the binding of VWF multimers to platelets, either increasing (type 2B) or decreasing (type 2M, 2A/2M) the affinity of VWF for platelets. In type 2A VWD, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding, impairing VWF secretion or increasing its susceptibility to proteolysis. Finally, a mutation localized in the carboxy-terminus CK domain, which is crucial for the dimerization of the VWF subunit, has been identified in a rare subtype 2A, formerly named IID.     

Classification and characterization of hereditary types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (unclassifiable) von Willebrand disease.

All variants of type 2 von Willebrand disease (VWD) patients, except 2N, show a defective von Willebrand factor (VWF) protein (on cross immunoelectrophoresis or multimeric analysis), decreased ratios for VWF:RCo/Ag and VWF:CB/Ag and prolonged bleeding time. The bleeding time is normal and FVIII:C levels are clearly lower than VWF:Ag in type 2N VWD. High resolution multimeric analysis of VWF in plasma demonstrates that proteolysis of VWF is increased in type 2A and 2B VWD with increased triplet structure of each visuable band (not present in types 2M and 2U), and that proteolysis of VWF is minimal in type 2C, 2D, and 2E variants that show aberrant multimeric structure of individual oligomers. VWD 2B differs from 2A by normal VWF in platelets, and increased ristocetine-induced platelet aggregation (RIPA). RIPA, which very likely reflects the VWF content of platelets, is normal in mild, decreased in moderate, and absent in severe type 2A VWD. RIPA is decreased or absent in 2M, 2U, 2C, and 2D, variable in 2E, and normal in 2N. VWD 2M is usually mild and characterized by decreased VWF:RCo and RIPA, a normal or near normal VWF multimeric pattern in a low resolution agarose gel. VWD 2A-like or unclassifiable (2U) is distinct from 2A and 2B and typically featured by low VWF:RCo and RIPA with the relative lack of high large VWF multimers. VWD type 2C is recessive and shows a characteristic multimeric pattern with a lack of high molecular weight multimers, the presence of one single-banded multimers instead of triplets caused by homozygosity or double hereozygosity for a mutation in the multimerization part of VWF gene. Autosomal dominant type 2D is rare and characterized by the lack of high molecular weight multimers and the presence of a characteristic intervening subband between individual oligimers due to mutation in the dimerization part of the VWF gene. In VWD type 2E, the large VWF multimers are missing and the pattern of the individual multimers shows only one clearly identifiable band, and there is no intervening band and no marked increase in the smallest oligomer. 2E appears to be less well defined, is usually autosomal dominant, and accounts for about one third of patients with 2A in a large cohort of VWD patients.     

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.     

An amino acid polymorphism in von Willebrand factor correlates with increased susceptibility to proteolysis by ADAMTS13

The hypothesis that increased ADAMTS13 (a disintegrin and metalloprotease with thrombospondin repeats) activity or increased susceptibility of von Willebrand factor (VWF) to proteolysis by ADAMTS13 may underlie type I von Willebrand disease (VWD) in some patients was investigated. Plasma from 4 patients with type I VWD was cryoprecipitated. ADAMTS13 activity in the VWF-poor cryodepleted fraction was assessed by incubation with purified VWF; susceptibility to proteolysis of the VWF in the VWF-rich cryoprecipitate was assessed by incubation with a normal, group O cryodepleted plasma. ADAMTS13 activity was similar in all 4 type I VWD cryodepleted plasmas and comparable to a normal control plasma. In contrast, the VWF of one patient showed increased susceptibility to proteolysis by ADAMTS13. Investigation of additional family members indicated that increased susceptibility was heritable, but it did not track uniquely with type I VWD. Sequence analysis of VWF exon 28 indicated that increased susceptibility to proteolysis tracked with the "G" allele of the A/G polymorphism at position 24/1282, encoding the amino acid polymorphism Tyr/Cys1584 ("G" = Cys1584). A prospective study of 200 individuals yielded 2 Tyr/Cys1584 heterozygotes; for both, plasma VWF showed increased susceptibility to proteolysis. The finding that an amino acid polymorphism in VWF may influence susceptibility to ADAMTS13 has potentially significant implications in diverse areas.     

Characterization of recessive severe type 1 and 3 von Willebrand Disease (VWD), asymptomatic heterozygous carriers versus bloodgroup O-related von Willebrand factor deficiency, and dominant type 1 VWD.

Recessive type 3 von Willebrand disease (VWD) is caused by homozygosity or double heterozygosity for two non-sense mutations (null alleles). Type 3 VWD is easy to diagnose by the combination of a strongly prolonged bleeding time (BT), absence of ristocetine-induced platelet aggregation (RIPA), absence of von Willebrand factor (VWF) protein, and prolonged activated partial thromboplastin time (aPTT) due to factor VIII:coagulant (FVIII:C) deficiency. VWD type 3 is associated with a pronounced tendency to mucocutaneous and musculoskeletal bleedings since early childhood. Carriers of one null allele are usually asymptomatic at VWF levels of 50% of normal. Recessive severe type 1 VWD is caused by homozygosity or double heterozygosity for a missense mutation. Recessive type 1 VWD differs from type 3 VWD by the presence of detectable von Willebrand factor: antigen VWF:Ag and FVIII:C levels between 0.09 and 0.40 U/mL. Patients with recessive type 1 VWD show an abnormal VWF multimeric pattern in plasma and/or platelets consistent with severe type 2 VWD. Carriers of a missense mutation may have mild bleeding and mild VWF deficiency and can be diagnosed by a double VWF peak on cross immunoelectrophoresis (CIE). There will be cases of mild and moderate recessive type 1 VWD due to double heterozygosity of two missense mutations, or with the combination of one missense mutation with a non-sense or bloodgroup O. Mild deficiency of VWF in the range of 0.20 to 0.60 U/mL, with normal ratios of von Willebrand factor: ristocetine cofactor/antigen VWF:RCo/Ag and VWF:collagen binding/antigen (VWF:CB/Ag), normal VWF multimers, and a completely normal response to desmopressin acetate (DDAVP) with VWF level rising from below to above 1.00 U/mL are very likely cases of so-called pseudo-VWF deficiency in individuals with normal VWF protein and gene. Autosomal dominant type 1 VWD variants are in fact type 2 variants caused by a heterozygous missense mutation in the VWF gene that produces a mutant VWF protein that has a dominant effect on normal VWF protein produced by the normal VWF allele with regard to the synthesis, processing, storage, secretion, and/or proteolysis of VWF in endothelial cells. A DDAVP challenge test clearly differentiates between dominant type 1 VWD phenotype and dominant type 2 M VWD.     

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.5 mol/l urea, and analyzed via multimer migration distance. The glutathione-S-transferase (GST) and histidine-tagged construct, E1554-R1668 of VWF (VWF115) was assayed via enzyme-linked immunosorbent assay: VWF115 was bound to anti-GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase-labelled anti-histidine tag antibody. All alterations examined in the Y1605-M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A-domain changes in VWF alter ADAMTS13-mediated proteolysis.     

An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers

Severe deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated into a high and a low group that differed by approximately 10 fold. Low ADAMTS13 activity was detected in mice containing 2 alleles of intracisternal A-type particle (IAP) retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified 2 truncated variants (IAP-a and IAP-b) in the low-activity mice. Both of the IAP variants lacked the 2 carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared with full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.     

Higher and lower active circulating VWF levels: different facets of von Willebrand disease

Most circulating von Willebrand factor (VWF) is normally inactive and incapable of binding platelets, but numerous disorders may modify the proportion of active VWF. We explored active VWF levels in patients with von Willebrand disease (VWD) whose VWF had a higher affinity for platelet glycoprotein (GP)Ib, but different susceptibilities to ADAMTS13 and multimer patterns (9 patients lacking large multimers, 10 with a normal pattern); 12 patients with VWF C2362F and R1819_C1948delinsS mutations, which make VWF resistant to ADAMTS13 were also studied. Type 2B patients with abnormal or normal multimers had significantly more active VWF (3·33 ± 1·6 and 3·74 ± 0·74, respectively; normal 0·99 ± 0·23). The type of VWF mutation influenced VWF activation: V1316M was associated with the highest levels in patients with abnormal multimers, and R1341W in those with normal multimers. Pregnancy induced gradually rising active VWF levels and declining platelet counts in one type 2B VWD patient without large multimers. Active VWF levels dropped significantly in patients homozygous for the C2362F mutation or heterozygous for R1819_C1948delinsS mutations (0·2 ± 0·03 and 0·23 ± 0·1, respectively), and less in cases heterozygous for the VWF C2362F mutation (0·55 ± 0·17). We demonstrate that VWF may be more or less activated, with or without any direct involvement of the A1 domain, and regardless of ADAMTS13.     

Platelet-VWF complexes are preferred substrates of ADAMTS13 under fluid shear stress

Endothelial cells secrete prothrombotic ultralarge von Willebrand factor (VWF) multimers, and the metalloprotease ADAMTS13 cleaves them into smaller, less dangerous multimers. This reaction is stimulated by tensile force applied to the VWF substrate, which may occur on cell surfaces or in the circulating blood. The cleavage of soluble VWF by ADAMTS13 was accelerated dramatically by a combination of platelets and fluid shear stress applied in a cone-plate viscometer. Platelet-dependent cleavage of VWF was blocked by an anti-GPIbalpha monoclonal antibody or by a recombinant soluble fragment of GPIbalpha that prevents platelet-VWF binding. Multimeric gel analysis showed that shear and platelet-dependent cleavage consumed large VWF multimers. Therefore, ADAMTS13 preferentially acts on platelet-VWF complexes under fluid shear stress. This reaction is likely to account for a majority of VWF proteolysis after secretion and to determine the steady-state size distribution of circulating VWF multimers in vivo.     

Variability in platelet‐ and collagen‐binding defects in type 2M von Willebrand disease

Type 2M von Willebrand disease (VWD) includes qualitative defects in von Willebrand factor (VWF) function, with normal multimer distribution but a defect in VWF activity with respect to platelet or collagen binding. We characterized novel VWF gene mutations found in type 2M VWD subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. Subjects were enrolled based on a pre‐existing diagnosis of type 2M VWD. Testing included full‐length gene sequencing, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding and multimer distribution. Recombinant VWF variants were synthesized using site‐directed mutagenesis and expressed in HEK293T cells. Platelet binding was measured by flow cytometry with fixed platelets and ELISA with recombinant glycoprotein Ibα (GPIbα). Four novel VWF A1 domain mutations were found in individuals with type 2M VWD: S1358N, S1387I, S1394F and Q1402P. All subjects had a history of bleeding, VWF:RCo < 40 IU dL^?1, VWF:RCo/VWF:Ag ratios <0.6 and normal multimer distribution. No defect in expression, secretion, or multimerization was found for any of the mutations. All showed decreased binding to intact platelets, and decreased or absent binding to a mutant GPIbα construct with spontaneous VWF binding. 1387I had decreased binding to all collagen types tested. 1402P had reduced binding exclusively to type VI collagen. Type 2M VWD is a heterogeneous category comprised of both collagen‐ and platelet‐binding defects. Understanding the precise defect for each mutation may ultimately lead to better diagnosis and treatment.     

Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD)

Type 1 von Willebrand disease (VWD) is characterized by a personal and family history of bleeding coincident with reduced levels of normal plasma von Willebrand factor (VWF). The molecular basis of the disorder is poorly understood. The aims of this study were to determine phenotype and genotype and their relationship in patients historically diagnosed with type 1 VWD. Families were recruited in 9 European countries based on previous type 1 VWD diagnosis. Bleeding symptoms were recorded, plasma phenotype analyzed, and VWF mutation analysis performed in all index cases (ICs). Phenotypic and molecular analysis stratified patients into those with or without phenotypes suggestive of qualitative VWF defects (abnormal multimers) and with or without mutations. A total of 105 of 150 ICs (70%) had mutations identified. A subgroup with abnormal multimers (38% of ICs, 57 of 150) showed a high prevalence of VWF gene mutations (95% of ICs, 54 of 57), whereas in those with qualitatively normal VWF, fewer mutations were identified (55% of ICs, 51 of 93). About one third of the type 1 VWD cases recruited could be reconsidered as type 2. The remaining group could be considered "true" type 1 VWD, although mutations were found in only 55%.     

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models

Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamic-expressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant ADAMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model.     

Reduced survival of type 2B von Willebrand factor, irrespective of large multimer representation or thrombocytopenia

Background

Type 2B von Willebrand factor (VWF) is characterized by gain of function mutations in the A1 domain inducing a greater affinity for platelet GPIb, possibly associated with the disappearance of large VWF multimers and thrombocytopenia.

Design and Methods

VWF survival was explored using 1-desamino-8-D-arginine vasopressin (DDAVP) in 18 patients with type 2B von Willebrand disease (VWD) and compared with their platelet count and large VWF multimer representation.

Results

A similarly significant shorter VWF survival, expressed as T_1/2elimination (T_1/2el), was observed in patients lacking large VWF multimers (type 2B) and in those with a normal multimer pattern (atypical type 2B) (4.47±0.41 h and 4.87±0.9 h, respectively, vs. normal 15.53±2.17 h) due mainly to a greater VWF clearance. The half-life of large VWF multimers, explored by VWF collagen binding (VWF:CB) activity, was likewise reduced. The similarly reduced VWF half-life was also confirmed by the increase in the VWF propeptide ratio (a useful tool for exploring VWF survival) which was found to be the same in type 2B and atypical type 2B patients. The post-DDAVP drop in platelet count occurred in all patients lacking large multimers but not in those with a normal multimer pattern. A correlation was always found between pre- and/or post-DDAVP thrombocytopenia and the lack of large VWF multimers in type 2B VWD while these were unrelated to the reduced VWF half-life.

Conclusions

In addition to demonstrating that a shorter VWF survival contributes to the type 2B and atypical type 2B VWD phenotype, our findings suggest that VWF clearance and proteolysis are independent phenomena.     

N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity

Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I-deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher K(m) but similar k(cat) value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.     

The prevalence of the cysteine1584 variant of von Willebrand factor is increased in type 1 von Willebrand disease: co-segregation with increased susceptibility to ADAMTS13 proteolysis but not clinical phenotype

The molecular pathogenesis of type 1 von Willebrand disease (VWD) is uncertain in most patients. We examined 30 type 1 VWD families in the UK Haemophilia Centre Doctors' Organization study. Heterozygosity for Y/C1584 was present in eight of 30 (27%) families and 19 of 76 (25%) individuals with type 1 VWD recruited into the study. Eighteen (95%) of these 19 individuals were blood group O. C1584 did not co-segregate with VWD in four families, and co-segregated in one family; the results were equivocal in three families. In all families increased susceptibility of von Willebrand factor (VWF) to a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) 13 proteolysis co-segregated with C1584 in affected and unaffected individuals. These data show that C1584, associated with blood group O, is prevalent among patients with type 1 VWD but not necessarily causative of disease and should not be used in isolation to diagnose VWD. Increased susceptibility of C1584 VWF to ADAMTS13 proteolysis may be physiologically significant and increase an individual's risk of bleeding and presenting with VWD.     

Insights into von Willebrand factor proteolysis: clinical implications

The proteolysis of von Willebrand factor (VWF) by the recently discovered metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin repeats), is a normal processing step in VWF biochemistry. Emerging data indicate that this step may be influenced by a variety of factors, some of which favour increased proteolysis and some of which compromise proteolysis. The former may predispose to bleeding, whilst the latter appears to be the underlying mechanism for thrombotic thrombocytopenic purpura (TTP). The new insights support the concept of "risk" in bleeding, particularly in the case of type 1 von Willebrand disease (VWD), in much the same way that risk is considered in venous thrombosis. This review presents relevant current knowledge of VWF proteolysis by ADAMTS13, and a novel model of how this may be implicated in type 1 VWD is proposed, based on events at the vessel wall at a time of haemostatic challenge.     

VWF and ADAMTS13 behavior in estradiol-treated HUVEC

Objectives: In this study, the role of 17β‐estradiol (E2) in the regulation of von Willebrand factor (VWF) and ADAMTS13 synthesis, storage, and secretion was investigated in cultured human umbilical vein endothelial cells (HUVEC). Methods: HUVEC were grown to 80–90% confluence and replaced with fresh medium containing E2 (1 nm ) or vehicle for 24 h, after which the supernatant medium and cell lysates were collected to measure VWF and ADAMTS13. VWF was evaluated by VWF:Ag and multimeric analysis. ADAMTS13 was evaluated by SDS‐PAGE. VWF and ADAMTS13 mRNA were quantified by real‐time PCR after E2 or vehicle exposure for 18 h. A functional effect of ADAMTS13 on HUVEC VWF protein synthesis was further evaluated using a short hairpin RNA (shRNA) to knockdown the expression of endogenous ADAMTS13. Results: E2 did not increase the release or intracellular VWF levels in HUVEC. However, E2 increased the production of intracellular ADAMTS13, although there was no evidence of significant effects of their release into culture medium. Incubation of HUVEC with E2 resulted in a significantly increased expression of VWF and ADAMTS13 mRNA. ADAMTS13 gene inactivation upregulates release and intracellular VWF levels in E2‐treated HUVEC. Conclusion: The results demonstrated that E2 may play a role in the regulation of VWF and ADAMTS13 gene expression and in its production in human endothelial cells. The mechanism of the protective effects of E2 on the cardiovascular system could be explained by the intracellular regulation of VWF produced by ADAMTS13.     

Shear stress-induced unfolding of VWF accelerates oxidation of key methionine residues in the A1A2A3 region

VWF is required for platelet adhesion to sites of vessel injury, a process vital for both hemostasis and thrombosis. Enhanced VWF secretion and oxidative stress are both hallmarks of inflammation. We recently showed that the neutrophil oxidant hypochlorous acid (HOCl) inhibits VWF proteolysis by ADAMTS13 by oxidizing VWF methionine 1606 (M1606) in the A2 domain. M1606 was readily oxidized in a substrate peptide, but required urea in multimeric plasma VWF. In the present study, we examined whether shear stress enhances VWF oxidation. With an HOCl-generating system containing myeloperoxidase (MPO) and H(2)O(2), we found that shear stress accelerated M1606 oxidation, with 56% becoming oxidized within 1 hour. Seven other methionine residues in the VWF A1A2A3 region (containing the sites for platelet and collagen binding and ADAMTS13 cleavage) were variably oxidized, one completely. Oxidized methionines accumulated preferentially in the largest VWF multimers. HOCl-oxidized VWF was hyperfunctional, agglutinating platelets at ristocetin concentrations that induced minimal agglutination using unoxidized VWF and binding more of the nanobody AU/VWFa-11, which detects a gain-of-function conformation of the A1 domain. These findings suggest that neutrophil oxidants will both render newly secreted VWF uncleavable and alter the largest plasma VWF forms such that they become hyperfunctional and resistant to proteolysis by ADAMTS13.