Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease.

Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type IIA von Willebrand disease. The patient's EC, compared with those from normal individuals, produced vWf that had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features characteristic of plasma vWf from patients with type IIA von Willebrand disease. The type IIA EC did produce a full spectrum of vWf multimers in both cell lysates and postculture medium, although the relative amounts of the largest species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites that contained approximately equal to 170-kDa proteolytic fragments, suggesting that this patient's functional defect is due to abnormal proteolysis and not to a primary failure of vWf subunit oligomerization. Moreover, the observed degradation appears to result from an abnormal vWf molecule and not elevated protease levels. These results suggest that this patient's von Willebrand disease phenotype is caused by increased proteolytic sensitivity of his vWf protein.     

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

Von Willebrand disease (VWD) is an inherited bleeding disorder, caused by quantitative (type 1 and 3) or qualitative (type 2) defects in von Willebrand factor (VWF). Gene therapy is an appealing strategy for treatment of VWD because it is caused by a single gene defect and because VWF is secreted into the circulation, obviating the need for targeting specific organs or tissues. However, development of gene therapy for VWD has been hampered by the considerable length of the VWF cDNA (8.4 kb [kilobase]) and the inherent complexity of the VWF protein that requires extensive posttranslational processing. In this study, a gene-based approach for VWD was developed using lentiviral transduction of blood-outgrowth endothelial cells (BOECs) to express functional VWF. A lentiviral vector encoding complete human VWF was used to transduce BOECs isolated from type 3 VWD dogs resulting in high-transduction efficiencies (95.6% +/- 2.2%). Transduced VWD BOECs efficiently expressed functional vector-encoded VWF (4.6 +/- 0.4 U/24 hour per 10(6) cells), with normal binding to GPIbalpha and collagen and synthesis of a broad range of multimers resulting in phenotypic correction of these cells. These results indicate for the first time that gene therapy of type 3 VWD is feasible and that BOECs are attractive target cells for this purpose.     

Unique expression of von Willebrand factor by type IIA von Willebrand''s disease endothelial cells

Endothelial cells (EC) were cultured from the umbilical cord of a male neonate whose mother was previously diagnosed with type IIA von Willebrand's disease (vWd). The diagnosis of type IIA vWd in the proband was confirmed by low ristocetin activity and the absence of the highest molecular weight (MW) forms of von Willebrand factor (vWf) in his platelet poor plasma. The vWf of EC cultured from the neonate's umbilical cord differed from that of control EC and the cell line EA.hy926 in two respects. Firstly, the full range of molecular weight forms was present in the patient EC lysate and, secondly, vWf:Ag expression was approximately seven-fold greater than that of control cells. Platelet lysates prepared from other affected members of the type IIA vWd family in the presence or absence of proteolytic inhibitors demonstrated a near normal vWf multimeric distribution. Resistance of these high MW forms to heat degradation was conferred by the presence of proteolytic inhibitors. Moreover, the full plasma vWf multimeric distribution could not be restored by the inclusion of EDTA. N-ethylmaleimide and leupeptin in the anticoagulant during the rapid preparation of platelet poor plasma. These findings lend support to the heterogeneous nature of type IIA vWd and has possible implications in the understanding of the intracellular processes involved in the biosynthesis and storage of the vWf macromolecular complex as well as the pathogenesis of type IIA vWd.     

Pharmacokinetics of von Willebrand factor and factor VIII coagulant activity in patients with von Willebrand disease type 3 and type 2

Nine patients with von Willebrand disease type 3, six with type 2B, one with type 2A, and one patient with type 1/2N were infused with one dose of ≈50 or 100 IU ristocetin cofactor activity (RCoF) per kg body weight of von Willebrand factor (vWF) (Human), a product with a very low content of factor VIII (FVIII). Blood samples were collected over 96 h. The data for RCoF and vWF antigen (vWF:Ag) were fitted to a 1-compartment model decay. The data for FVIII:C were fitted to a model with a linear time 'synthesis' term and a 1-compartment decay. Results in von Willebrand disease type 3 patients (nine patients; 10 infusions) indicated a volume of distribution of 39.9 and 39.8 mL kg⁻¹ for RCoF and vWF:Ag, respectively. The FVIII:C rate of synthesis was 6.4 U dL⁻¹ h⁻¹ (range: 4.4–8.8). The decay rates for FVIII:C, RCoF, and vWF:Ag were 0.041 (h⁻¹) [ t _1/2: 16.9 h]; 0.061 (h⁻¹) [ t _1/2: 11.3 h] and 0.006 (h⁻¹) [ t _1/2: 12.4 h], respectively. In patients with von Willebrand disease type 2 ( n  = 8) the RCoF mean volume of distribution was 46 mL kg⁻¹. The factor VIIIC mean rate of synthesis was 5.5 U dL⁻¹h⁻¹ and the decay rate 0.043 (h⁻¹) [ t _1/2: 16.1 h]. The rate of decay for RCoF and vWF:Ag were 0.050 (h⁻¹) [ t _1/2: 13.9 h] and 0.044 (h⁻¹) [ t _1/2: 15.7 h], respectively.     

Heterogeneity of type I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor

Type I von Willebrand disease (vWD) is characterized by equally low plasma concentrations of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RiCof) and by the presence of all vWF multimers in sodium dodecyl sulfate (SDS)-agarose gel electrophoresis. For 17 patients (13 kindreds) diagnosed with these criteria, we have studied the platelet contents of vWF:Ag and RiCof and the changes of these in plasma after DDAVP infusion. Platelet vWF:Ag and RiCof were normal in four kindreds (called "platelet normal" subgroup); following 1-deamino- 8-D-arginine vasopressin; plasma vWF:Ag, RiCof and the bleeding time (BT) became normal. In six kindreds, platelet vWF:Ag and RiCof were equally low (platelet low); after DDAVP, plasma vWF:Ag and RiCof remained low, and the BT was prolonged. In three additional kindreds, platelets contained normal concentrations of vWF:Ag, but RiCof was very low (platelet discordant); even though a complete set of multimers was found in plasma and platelets, there was a relatively small amount of large multimers. After DDAVP, plasma vWF:Ag became normal, but RiCof remained low and the BT was very prolonged. These findings demonstrated that there can be an abnormal vWF (RiCof less than vWF:Ag) even in type I vWD, coexisting with a complete set of vWF multimers (platelet discordant); that the abnormal vWF can be shown more clearly in platelets than in plasma or else in plasma after DDAVP infusion; and that DDAVP normalizes the BT only in those patients with normal platelet levels of both vWF:Ag and RiCof (platelet normal).     

Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF-treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.     

Hyper-responsiveness to DDAVP for patients with type I von Willebrand's disease and normal intra-platelet von Willebrand factor

Desmopressin (DDAVP) has gained wide acceptance as the drug of first choice in the treatment or prevention of haemorrhages in the majority of patients with von Willebrand's disease (vWd). However, data concerning the clinical effectiveness of DDAVP refer generally to mild vWd, with factor VIII and vW factor levels usually above 20% of normal. In 14 patients with type I vWd characterized by very low plasma levels of factor VIII coagulant activity (VIII:C) and vWf, measured as ristocetin cofactor activity (lower than 20% and 3% of normal respectively), but with a normal intraplatelet content of vWf, a test infusion of DDAVP (0.4 microgram/kg) elicited a very marked increase of VIII:C and vWf and normalized the bleeding time. All these patients subsequently underwent tooth extraction after DDAVP infusion. The incidence of bleeding was remarkably low, with only two minor late bleeding episodes easily stopped by repeating DDAVP infusion. Compared to the cases of type I vWd with unknown intraplatelet vWf content reported in the literature, this subgroup of patients had a more marked, albeit short-lived, increment of VIII:C and vWf.     

Molecular characterization of Iranian patients with type 3 von Willebrand disease

Summary.   von Willebrand's disease (VWD) type 3 is a rare but severe autosomal-recessive inherited bleeding disorder with a prevalence higher in certain locations where consanguineous marriages are relatively frequent. The genetic defects causing recessive type 3 VWD in 10 unrelated families from Iran have been investigated and the genetic heterogeneity among these patients was evaluated. All exons and their flanking regions of von Willebrand factor gene were amplified by PCR and sequenced using specific primers. Eight patients were fully characterized at the molecular level. Six different gene alterations were identified. All the mutations caused null alleles, three being nonsense mutations (Q104X, Q793X and E1981X), two possible splice site mutations (2443-1G>C and 1110-1G>A) and one small deletion (3237delA). Three of them have not been described previously. Most patients were born from consanguineous marriages and all were homozygous for their mutations. The results confirm that molecular defects in type 3 VWD are heterogeneous with mutations arising randomly within the entire gene.     

Increased release of von Willebrand factor antigen from endothelial cells by anti-DNA autoantibodies.

OBJECTIVE--To determine whether antibodies to double stranded DNA (anti-dsDNA) have a pathogenic role in systemic lupus erythematosus (SLE). METHODS--IgG was purified from 17 patients with SLE (median anti-dsDNA titre 1212 IU/ml) and nine healthy controls (median titre 40 IU/ml). Anti-dsDNA depleted polyclonal IgG (median anti-dsDNA titre 17 IU/ml) was also prepared from sera of the 17 patients by affinity chromatography on a DNA cellulose column. Binding to antiendothelial cell antibodies (AECA) and expression of von Willebrand factor (VWF) antigen by cultured human umbilical vein endothelial cells (HUVECs) were studied by flow cytometry. RESULTS--The percentage of HUVECs binding to AECA or expressing VWF was greater for cells incubated with IgG from patients with SLE than for cells incubated with control IgG, though values did not reach statistical significance; nevertheless, HUVECs incubated with IgG from patients expressed a greater mean fluorescence intensity with AECA (p = 0.0001) and greater VWF expression (p = 0.019). Both the fluorescence intensity and percentage of HUVECs binding to AECA or expressing VWF were significantly greater in HUVEC incubated with IgG containing anti-dsDNA than in those incubated with anti-dsDNA depleted IgG. The concentration of VWF in the supernatant was significantly increased in HUVECs incubated with IgG containing anti-dsDNA compared with control IgG or anti-dsDNA depleted IgG. Pretreatment of HUVECs with native DNA before incubation with IgG from lupus patients did not increase binding to AECA, or expression or release of VWF. CONCLUSIONS--Our study provides in vitro evidence that antibodies to DNA have a pathogenic role in the induction of inflammatory injury of the vascular endothelium in SLE.     

Characterization of von Willebrand factor gene defects in two unrelated patients with type IIC von Willebrand disease

Genetic studies were performed in two unrelated patients with the IIC phenotype of von Willebrand disease (vWD) characterized by the increased concentration of the protomeric form of von Willebrand factor (vWF). In patient B, the sequencing of both exons 15 and 16 of the vWF gene showed two sequence alterations: a 3-bp insertion in exon 15 resulting in the insertion of a Glycine at position 625 (625insGly) and a 2-bp deletion in exon 16 leading to a premature translational stop at codon 711 (711 ter), at the heterozygote state. Patient A was found homozygous for a single point mutation also localized in exon 15 and responsible for the substitution Cys623Trp. These candidate mutations were not found in a panel of 96 normal chromosomes, suggesting a causal relationship with IIC vWD phenotypic expression. The composite heterozygote or homozygote state of both patients supports the recessive mode of inheritance already described for this phenotype. Furthermore, the localization of these gene defects in the D2 domain of vWF propeptide, known to play an important role in vWF multimerization, provides another argument in favor of their causative effect regarding the peculiar multimeric pattern of vWF in these patients.     

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.     

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease

To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312-19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD.     

Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells

A case of familial Pelger-Huet anomaly in a 3-year-old boy with acute lymphoblastic leukaemia is described. This unique association was investigated through trial observations of the peripheral blood smear and bone marrow obtained during the child's treatment with chemotherapy. The average lobe index (ALI) of neutrophils was 42 with no three-lobed forms at the time of the initial diagnosis. During antimetabolite maintenance therapy with 6-mercaptopurine and methotrexate the ALI was 1.87 and three-lobed forms were present. The behaviour of the P-HA cells to heat induced radial hypersegmentation of the nucleus was examined in other family members. The mechanism by which heat and folate deficiency induce neutrophil segmentation is preserved in the familial Pelger-Huet anomaly.     

Type I von Willebrand disease, subtype 'platelet low': decreased platelet adhesion can be explained by low synthesis of von Willebrand factor in endothelial cells

Summary Endotheial cells (EC) were isolated from the umbilical vein of a newborn girl with type I 'platelet low' von Willebrand disease (I vWD) and endothelial localization and release and the ability of subendothelial von Willebrand factor (vWF) to support platelet adhesion were compared with those of normal EC. vWF was detectable by immunofluorescence in Weibel-Palade bodies, but the number of Weibel-Palade bodies positive for vWF was lower than in control EC. Patient EC released into the medium significantly smaller amount of vWF, both constitutively and after their stimulation. The vWF content of the extracellular matrix of patient EC was 38% that of control EC matrix. Platelet adhesion studies were performed under flow conditions with umbilical arteries and EC matrices of cultured EC. Using normal citrated whole blood as perfusate, platelet adhesion was lower in the umbilical artery of the patient (9 ± 1% v 35 ± 4% for the control) and in her EC matrix (7 ± 1% v 21 ± 2% of control). When patient EC matrix was perfused with vWF-deficient reconstituted blood, adhesion was 17 ± 3% v 32 ± 3% for control EC matrix; preincubation of patient EC matrix with 1 U/ml vWF increased the adhesion to 30 ± 6%. These data establish that low contents of vWF in EC and subendothelium are important characteristics of type I vWD 'platelet low', and that such characteristics correlate with low platelet adhesion to the subendothelium.     

Basal secretion of von Willebrand factor from human endothelial cells

Endothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.     

Molecular defects in type 3 von Willebrand disease: updated results from 40 multiethnic patients

Type 3 von Willebrand disease (VWD) is characterized by unmeasurable von Willebrand factor (VWF) levels in plasma and platelets and severe hemorrhagic symptoms. We have characterized at the molecular level a group of 40 patients (12 Italians, 14 Iranians, and 14 Indians) to evaluate genetic heterogeneity among these populations. Some of these patients have been previously investigated by us (mutations shown in italics); they are included in this study to provide a more comprehensive pattern of gene defects in type 3 VWD. Patients' DNA were first tested for more frequently reported mutations, then screened by single-strand conformation polymorphism and direct sequence analysis. Fifty gene defects were identified, of which 45 are novel. As expected most of these defects caused null alleles, 17 being nonsense mutations (Q218X, W222X, R365X, R373X, Y610X, W642X, E644X, Q706X, Q1311X, S1338X, Q1346X, Y1542X, R1659X, E1981X, E2129X, R2434X, and Q2544X), 12 small deletions (191delG, 276delT, 788del24, 2016del4, 2157delA, 2269delCT, 2435delC, 4092delAC, 6182delT, 7294delGT, 7683delT, and 8241del9), 4 small insertions (4414insC, 7130insC, 7137insT, and 7674insC), 8 possible splice site mutations (1110(-1)G-->A, 1946(-4)C-->T, 3108(+5)G-->A, 3379(+1)G-->A, 5053(+1)G-->A, 5170(+10)C-->T, 6977(-1)G-->C, and 7729(+7)C-->T), 8 candidate missense mutations (D47H, S85P, D141N, D141Y, C275S, C1071F, C2174G, and C2804Y), and 1 large gene deletion (exons 23-52). Only 2 of these patients have developed alloantibodies against VWF. This study extend our previous finding that mutations responsible for type 3 VWD are scattered throughout the entire VWF gene and that there is no founder effect in these three populations studied.     

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.     

Irradiation induces release of von Willebrand protein from endothelial cells in culture

Human umbilical vein endothelial cells in tissue culture were irradiated with doses between 0 and 40 Gy, and the released von Willebrand (vW) protein and that which remained associated with the cells was quantitated. Doses of 20 Gy and higher produced a statistically significant increase in amount of vW protein secreted. This release was present whether the cells were labeled continuously throughout the experiment or just prelabeled before irradiation. An increase in fibronectin secretion was not observed. The release response to radiation was slow, reaching significance close to 24 hours after irradiation. The release of vW protein was not due to cell lysis, because the secreted vW protein contained very little of the large 260- kilodalton vW precursor subunit present in cell lysates and the cells appeared intact by immunofluorescence staining.     

Response of von Willebrand factor parameters to desmopressin in patients with type 1 and type 2 congenital von Willebrand disease: diagnostic and therapeutic implications

In the present study, we prospectively evaluated the contribution of the von Willebrand factor collagen-binding activity (vWF:CBA) assay, vWF multimeric analysis, and the response to intravenous desmopressin (DDAVP) to correctly diagnose and classify congenital von Willebrand disease (CvWD) in 24 probands with mild to moderate type 1 vWD, 6 probands with severe CvWD type 1, and 12 probands with type 2 CvWD. CvWD type 1 of mild to moderate severity is featured by proportionally decreased levels of vWF antigen (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCof), and vWF:CBA between 0.20 and 0.60 u/mL and a normal response to DDAVP of factor (F) VIIIc and all vWF parameters. Severe type 1 CvWD with vWF parameters below 0.10 or 0.20 u/mL is associated with a decreased response to DDAVP of all vWF parameters, indicating a defective synthesis or secretion vWF by endothelial cells, or both. CvWD 2M may present as severe type 1 CvWD, as type 1 "platelet-discordant" CvWD, or with the combination of a discrepant vWF:RCof/Ag ratio and the presence of all vWF multimers. Ristocetin-induced platelet aggregation (RIPA) is normal in type 1 CvWD. CvWD 2M is typically featured by decreased RIPA, normal or near normal vWF multimers, and no or only a poor response to DDAVP of vWF:RCof as compared with a fairly good response to DDAVP of vWF:Ag and vWF:CBA. CvWD Vicenza is characterized by unusually large vWF multimers and very low levels of FVIIIc, vWF:Ag, and vWF:RCof. CvWD Vicenza differs from CvWD 2M because the vWF:RCof/Ag ratios are completely normal before and after DDAVP; the response to DDAVP is equally good for FVIIIc, vWF:Ag, vWF:RCof, and vWF:CBA and is followed by very short half-life times for FVIIIc and all vWF parameters. Pertinent findings in type 2A and 2B CvWD included prolonged Ivy bleeding time (BT), low vWF:RCof/Ag and vWF:CBA ratios, absence of the high vWF multimers, and, depending on the severity of the absence of intermediate vWF multimers, pronounced increase of low vWF multimers and vWF degradation products because of increased proteolysis of the high and intermediate vWF multimers. RIPA is normal in CvWD 2A and increased in CvWD 2B. The response to DDAVP in CvWD 2A is normal for FVIIIc and vWF:Ag but is transient with partial correction and short half-life times of vWF:CBA and vWF:RCof. DDAVP does not correct BT and multimeric patterns in CvWD type 2B, despite significant increase of vWF parameters. CvWD types 2C, 2D, and 2E are featured by very low functional vWF parameters, the presence of typically abnormal vWF multimers, a very poor response of vWF:CBA, a decreased response of vWF:RCof, and a fairly good response of vWF:Ag to DDAVP with no correction of prolonged Ivy BT and no correction of the vWF multimeric pattern as the consequence of a multimerization or dimerization defect of the vWF molecules. CvWD type 2N usually presents with much lower levels for FVIIIc as compared with vWF, normal Ivy BT, and normal vWF multimeric pattern. The response to DDAVP is normal for all vWF parameters but is decreased for FVIIIc with shortened half-life times.