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Competing Interests: Declaration of competing interest There are no competing interest to disclose.

Background: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions., Objective: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS., Methods: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay., Results: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14)., Conclusions: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used., Competing Interests: Declaration of competing interest statement J.E. received research support from CSL Behring outside the scope of this project and fees for educational activities from Roche and Celgene, which fees go to the institution. J.G. in the last 3 years received fees for lectures or consultancy from LFB outside the scope of this work. F.L. received unrestricted research grants from CSL Behring, Takeda, SOBI, and uniQure. He is a consultant for CSL Behring, Takeda, BioMarin, and uniQure, of which the fees go to the University. He has been a DSMB member of a study sponsored by Roche. P.M.M. is member of the scientific board for the Bayer Awards. Speaker fee from Bayer and Kedrion, Roche for lectures at educational symposia. C.S., in the last 3 years, has received honoraria for consulting or speaker bureau from Bayer, CSL Behring, Takeda, Novonordisk, Sobi, Novartis, Pfizer, Amgen, and Roche. A.T. received research support or honoraria for lectures or consultancy from CSL Behring, Octapharma, and Takeda outside of this work. F.P. reported participation at advisory board of Sanofi, Sobi, Takeda, Roche, and BioMarin. A.B.F. reported advisory board participation of CSL Behring, Grifols, Takeda, Octapharma, LFB, and Kedrion. J.O. has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda; consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin, Biotest, Chugai Pharmaceutical Co., Ltd., CSL Behring, Freeline, Grifols, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd., Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. GC reported advisory board participation/speaker fee of CSL Behring, Grifols, Takeda, LFB, and Kedrion. The remaining authors declare no competing financial interests., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Plasma-derived von Willebrand factor (VWF) (Wilfactin ® , LFB, France) was developed for prophylaxis and treatment of haemorrhages in both adults and adolescents with von Willebrand disease (VWD). Replacement therapy in paediatric patients is a key element of the clinical trial programme., Material and Methods: Patients aged <6 years with severe VWD were enrolled in a multinational, open-label study to evaluate the in vivo recovery for Wilfactin ® , and its efficacy in preventing and treating bleeding episodes and during surgery. Overall haemostatic efficacy based on a 4-point scale was assessed by investigators. The treatment period ≥18 months investigated the long-term safety., Results: Nine patients, including 7 with type 3 VWD were exposed to treatment with Wilfactin ® for up to 4.2 years. Recovery of VWF in 7 patients (n=5 type 3, n=1 type 2, n=1 type 1) was 1.8±0.4 IU/dL per IU/kg. Of the 62 bleeds, 89% were controlled with one (73%) or two (16%) infusions of Wilfactin ® . The median dose per infusion was 54 IU/kg. A factor VIII dose was co-administered in 1.6% of bleeds. "Excellent"/"Good" haemostatic efficacy was achieved in 90.3% of episodes. Six patients underwent 11 minor surgical interventions. Treatment duration was 1 day (range: 1-6 days) with a dose administered 30-60 minutes before procedure of 56 IU/kg (range: 41-106 IU/kg). Haemostasis was rated as "Excellent" in all surgeries. During 4-year prophylactic treatment in one patient, breakthrough bleeds were reported in 2.2% of infusions. No VWF inhibitors, thromboembolic events or allergic/anaphylactic-type reactions were observed following a total exposure of 770 days., Discussion: The results show that Wilfactin ® provides a safe and effective treatment in patients <6 years of age with severe VWD.

Background: Type 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag., Objective: To investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity., Methods: European and Iranian type 3 patients were enrolled in the 3WINTERS-IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients' diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann-Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges-Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman's rank correlation., Results: Homozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants ([VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2-2.7; P = .016]; [VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0-4.2; P = .054])., Fviii: C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778)., Conclusions: An increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Von Willebrand disease (VWD) is one of the most common inherited bleeding disorders, imposing a substantial health impact and financial burden. The C ost of v on Willebrand disease in E urope: A S ocioeconomic S tudy (CVESS) characterises the socio-economic cost of VWD across Germany, Spain, Italy, France, and the UK., Methods: A retrospective, cross-sectional design captured 12 months of patient disease management, collected from August-December 2018, for 974 patients. This enabled estimation of direct medical, direct non-medical and indirect costs, utilising prevalence estimates to extrapolate to population level., Results: Total annual direct medical cost (including/excluding von Willebrand factor [VWF]) across all countries was the highest cost (€2 845 510 345/€444 446 023), followed by indirect costs (€367 330 271) and direct non-medical costs (€60 223 234). Differences were seen between countries: the UK had the highest direct medical costs excluding VWF (€159 791 064), Italy the highest direct-non medical (€26 564 496), and Germany the highest indirect cost burden (€197 036 052). Total direct medical costs per adult patient increased across VWD types with Type 1 having the lowest cost (€23 287) and Type 3 having the highest cost (€133 518)., Conclusion: A substantial financial burden arises from the prevalence of VWD for the European healthcare systems considered.

Type 3 von Willebrand disease (VWD3) is a rare and severe bleeding disorder characterized by often undetectable von Willebrand factor (VWF) plasma levels, a recessive inheritance pattern, and heterogeneous genotype. The objective of this study was to identify the VWF defects in 265 European and Iranian patients with VWD3 enrolled in 3WINTERS-IPS (Type 3 Von Willebrand International Registries Inhibitor Prospective Study). All analyses were performed in centralized laboratories. The VWF genotype was studied in 231 patients with available DNA (121 [115 families] from Europe [EU], and 110 [91 families] from Iran [IR]). Among 206 unrelated patients, 134 were homozygous (EU/IR = 57/77) and 50 were compound heterozygous (EU/IR = 43/7) for VWF variants. In 22 patients, no or only one variant was found. A total of 154 different VWF variants (EU/IR = 101/58 [5 shared]) were identified among the 379 affected alleles (EU/IR = 210/169), of which 48 (EU/IR = 18/30) were novel. The variants p.Arg1659*, p.Arg1853*, p.Arg2535*, p.Cys275Ser, and delEx1&#95;Ex5 were found in both European and Iranian VWD3 patients. Sixty variants were identified only in a single allele (EU/IR = 50/10), whereas 18 were recurrent (≥3 patients) within 144 affected alleles. Nine large deletions and one large insertion were found. Although most variants predicted null alleles, 21% of patients carried at least 1 missense variant. VWD3 genotype was more heterogeneous in the European population than in the Iranian population, with nearly twice as many different variants. A higher number of novel variants were found in the Iranian VWD3 patients., (© 2021 by The American Society of Hematology.)

Background: Despite a high prevalence of angiodysplasia, no specific guidelines are available for the modalities of endoscopic exploration of gastrointestinal (GI) bleeding in von Willebrand disease (VWD). Whether VWD patients could benefit from video capsule endoscopy (VCE) looking for angiodysplasia eligible to endoscopic treatment or at high risk of bleeding is unknown., Objectives: To assess the diagnostic efficacy for angiodysplasia and the prognostic value of VCE on top of conventional endoscopy in VWD patients with GI bleeding., Patients/methods: A survey was sent to the 30 centers of the French-network on inherited bleeding disorders to identify VWD patients referred for endoscopic exploration of GI bleeding from January 2015 to December 2017. Data obtained included patient characteristics, VWD phenotype/genotype, GI bleeding pattern, results of endoscopic investigations, and medical management applied including endoscopic therapy. We assessed by Kaplan-Meier analysis the recurrence-free survival after the first GI bleeding event according to endoscopic categorization and, in patients with angiodysplasia, to the presence of small-bowel localizations on VCE exploration., Results: GI bleeding source localization was significantly improved when including VCE exploration (P < .01), even in patients without history of angiodysplasia (P < .05). Patients with angiodysplasia had more GI bleeding recurrences (P < .01). A lower recurrence-free survival was observed in patients with angiodysplasia (log-rank test, P = .02), and especially when lesions were located in the small bowel (log-rank test, P < .01), even after endoscopic treatment with argon plasma coagulation (log-rank test, P < .01)., Conclusion: VCE should be more systematically used in VWD patients with unexplained or recurrent GI bleeding looking for angiodysplasia eligible to endoscopic treatment or at high risk of relapse., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Type 3 von Willebrand's disease (VWD) patients present markedly reduced levels of von Willebrand factor and factor VIII. Because of its rarity, the bleeding phenotype of type 3 VWD is poorly described, as compared to type 1 VWD., Aims: To evaluate the frequency and the severity of bleeding symptoms across age and sex groups in type 3 patients and to compare these with those observed in type 1 VWD patients to investigate any possible clustering of bleeding symptoms within type 3 patients., Methods: We compared the bleeding phenotype and computed the bleeding score (BS) using the MCMDM-1VWD bleeding questionnaire in patients enrolled in the 3WINTERS-IPS and MCMDM-1VWD studies., Results: In 223 unrelated type 3 VWD patients, both the BS and the number of clinically relevant bleeding symptoms were increased in type 3 as compared to type 1 VWD patients (15 versus 6 and 5 versus 3). Intracranial bleeding, oral cavity, hemarthroses, and deep hematomas were at least five-fold over-represented in type 3 VWD. A more severe bleeding phenotype was evident in patients having von Willebrand factor antigen levels < 20 IU/dL at diagnosis in the two merged cohorts. In type 3 patients, there was an apparent clustering of hemarthrosis with gastrointestinal bleeding and epistaxis, whereas bleeding after surgery or tooth extraction clusters with oral bleeding and menorrhagia., Conclusions: In the largest cohort of type 3 VWD patients, we were able to describe a distinct clinical phenotype that is associated with the presence of a more severe hemostatic defect., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: A triple-secured plasma-derived von Willebrand factor (pdVWF) almost devoid of factor VIII (FVIII):WILFACTIN ® , was approved in France in 2003, and then in other countries for the treatment of patients with von Willebrand disease (VWD)., Objective: To investigate long-term safety and efficacy of the product in real-life over the first 5 post-approval years., Patients/methods: This prospective, observational, national post-marketing study (PMS) enrolled patients of all ages and VWD types. Patients were observed for up to 3 years and treated for one or more occasions. Efficacy was assessed for each major event. Breakthrough bleeding rate 3 days post-infusion and annualized bleeding rate (ABR) were also evaluated for long-term prophylaxis., Results: Overall, 155 of 174 patients enrolled from 31 centers were eligible for efficacy assessment. Most patients (76.8%) were severely affected (VWF:RCo ≤ 15 IU/dL). They were treated for 743 bleeds and 140 surgeries including childbirth. Efficacy outcomes were excellent/good for 98.2% of 56 major surgeries and 94.0% of 67 major bleeds. Approximately 75% of 49 major mucosal bleeds were effectively managed without FVIII co-administration. In 32 patients receiving prophylaxis, breakthrough bleeding occurred in 1.5% of infusions and median ABR was 1.0 for 20 patients treated ≥ 12 months. Excellent tolerability was confirmed with no safety concerns. No thrombotic events were observed., Conclusions: Results from this PMS increase the clinical experience of a FVIII-poor pdVWF in patients of all ages and VWD types including those with thrombotic risk factors and emphasize that giving FVIII is not always mandatory to effectively treat patients with severe VWD., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion., (© 2020 by The American Society of Hematology.)

Von Willebrand disease-type 2A (VWD-2A) and acquired von Willebrand syndrome (AVWS) due to aortic stenosis (AS) or left ventricular assist device (LVAD) are associated with an increased proteolysis of von Willebrand factor (VWF). Analysis of VWF multimeric profile is the most sensitive way to assess such increased VWF-proteolysis. However, several technical aspects hamper a large diffusion among routine diagnosis laboratories. This makes early diagnosis and early appropriate care of increased proteolysis challenging. In this context of unmet medical need, we developed a new ELISA aiming a quick, easy and reliable assessment of VWF-proteolysis. This ELISA was assessed successively in a LVAD-model, healthy subjects (n=39), acquired TTP-patients (n=4), VWD-patients (including VWD-2A(IIA), n=22; VWD-2B, n=26; VWD-2A(IIE), n=21; and VWD-1C, n=8) and in AVWS-patients (AS, n=9; LVAD, n=9; and MGUS, n=8). A standard of VWF-proteolysis was specifically developed. Extent of VWF-proteolysis was expressed as relative percentage and as VWF proteolysis/VWF:Ag ratio. A speed-dependent increase in VWF-proteolysis was assessed in the LVAD model whereas no proteolysis was observed in TTP-patients. In VWD-patients, VWF-proteolysis was significantly increased in VWD-2A(IIA) and VWD-2B and significantly decreased in VWD-2A(IIE) versus controls (p< 0.0001). In AVWS-patients, VWF-proteolysis was significantly increased in AS- and LVAD-patients compared to controls (p< 0.0001) and not detectable in MGUS-patients. A significant increase in VWF-proteolysis was detected as soon as three hours after LVAD implantation (p< 0.01). In conclusion, we describe a new ELISA allowing a rapid and accurate diagnosis of VWF-proteolysis validated in three different clinical situations. This assay represents a helpful alternative to electrophoresis-based assay in the diagnosis and management of AVWS with increased VWF-proteolysis.

von Willebrand disease (VWD) is a genetic bleeding disease due to a defect of von Willebrand factor (VWF), a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. VWD include quantitative defects of VWF, either partial (type 1 with VWF levels <50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50 IU/dL). The French reference center for VWD performed a laboratory phenotypic and genotypic analysis in 1167 VWD patients (670 families) selected by their basic biologic phenotype: type 3, type 2, and type 1 with VWF levels <30 IU/dL. In these patients indeed, to achieve an accurate diagnosis of VWD type and subtype is crucial for the management (treatment and genetic counseling). A phenotype/genotype correlation was present in 99.3% of cases; 323 distinct VWF sequence variations (58% of novel) were identified (missense 67% versus truncating 33%). The distribution of VWD types was: 25% of type 1, 8% of type 3, 66% of type 2 (2A: 18%, 2B: 17%, 2M: 19%, 2N: 12%), and 1% of undetermined type. Type 1 VWD was related either to a defective synthesis/secretion or to an accelerated clearance of VWF. In type 3 VWD, bi-allelic mutations of VWF were found in almost all patients. In type 2A, the most frequent mechanism was a hyper-proteolysis of VWF. Type 2B showed 85% of patients with deleterious mutations (distinct from type 2B New York). Type 2M was linked to a defective binding of VWF to platelet glycoprotein Ib or to collagen. Type 2N VWD included almost half type 2N/3. This biologic study emphasizes the complex mechanisms for both quantitative and qualitative VWF defects in VWD. In addition, this study provides a new epidemiologic picture of the most bleeding forms of VWD in which qualitative defects are predominant.

We evaluated the use of the turbidimetric HemosIL von Willebrand Factor (VWF) Activity assay (VWF:Act) on the STA-R automated coagulometer (Stago, Asnières, France) for the diagnosis of von Willebrand disease (VWD). For this, we prospectively screened 268 patients. As a second part, we retrospectively assayed 111 patients with well-defined VWD subtype. In the first prospective study, we demonstrate that in most cases of VWD, VWF ristocetin cofactor activity (VWF:RCo) and VWF:Act are highly correlated but that they both cannot be considered a good screening assay when used alone, since they could miss about 25% of VWF abnormalities. However, the association of VWF:Act analysis and the Platelet Function Analyzer-100 (PFA-100) test constitutes an excellent screening strategy. In our second retrospective study concerning VWD subtypes, VWF:RCo and VWF:Act were well correlated but could be very discrepant, especially for some cases of type 2M VWD. We consider that VWF:RCo remains the "reference assay" for VWD subtype classification.

The relationships between the Platelet Function Analyzer (PFA)-100 and von Willebrand factor (VWF) levels and bleeding score (BS) were evaluated within a multicentre project on Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand disease (MCMDM-1VWD). PFA-100 closure time, either with epinephrine (EPI) or adenosine diphosphate (ADP)-cartridges, was measured in 107 index cases, 105 affected and 71 unaffected family members, and 79 healthy controls. By regression analysis VWF levels were strongly related to both closure times, with a non-linear progression. In a multiple stepwise regression model, age- and sex-adjusted PFA-100 ADP and VWF ristocetin cofactor activity (VWF:RCo) were independently associated with BS. Most of the variation of BS was predicted by PFA-100 ADP and VWF:RCo alone. In the subgroup of patients with subtle abnormalities of the multimeric pattern, VWF was invariably reduced and closure time prolonged in almost all of them. Neither PFA-100 ADP nor EPI closure times appeared to significantly improve the diagnostic capability of VWF antigen (VWF:Ag) measurement. Thus, in an unselected population a normal PFA-100 would be useful to exclude VWD, but whether it could replace the more specific VWF assay in patients with significant mucocutaneous bleeding symptoms remains to be investigated prospectively., (© 2010 Blackwell Publishing Ltd.)

Background: von Willebrand factor (VWF) variant c.2771G>A; p.R924Q has been described as a benign polymorphism or a possible marker for a null allele and been associated with mild bleeding phenotypes. It was identified in several patients in recent type 1 von Willebrand disease (VWD) studies., Objectives: To determine whether the p.R924Q allele contributes to reduced VWF levels and type 1 VWD., Methods: One thousand one hundred and fifteen healthy controls and 148 index cases from the MCMDM-1VWD study were genotyped for c.2771G>A; VWF and FVIII levels were analyzed in ABO blood group stratified individuals and the p.R924Q variant was expressed in 293 EBNA cells., Results: c.2771G>A was present in six index cases, five of whom had a second VWF variant which probably contributed to the phenotype. A common core haplotype identified in families, which included the rare G allele of c.5843-8C>G, was present in the majority of 35 c.2771G>A heterozygous controls. c.2771G>A contributed about 10% variance in VWF and FVIII levels in controls and 35% variance when co-inherited with blood group O. Recombinant p.R924Q VWF had no effect on in vitro expression and heterozygous family members had normal VWF-FVIII binding and normal clearance of VWF and FVIII., Conclusions: The allele bearing c.2771A leads to reductions in VWF and FVIII levels particularly in combination with blood group O. Its inheritance alone may be insufficient for VWD diagnosis, but it appears to be associated with a further VWF level reduction in individuals with a second VWF mutation and it contributes to population variance in VWF and FVIII levels., (© 2010 International Society on Thrombosis and Haemostasis.)

Although von Willebrand disease (VWD) is now well-described, many facets of diagnosis and management continue to be debated. The diagnosis of type 1 disease can be difficult but recent genetic analyses help to distinguish many factors which can influence von Willebrand factor (VWF) levels and bleeding phenotype. Type 2 disease (functional abnormalities) includes a particularly interesting group of disorders with faulty binding between VWF and FVIIIC (Normandy) where treatment methods need careful consideration. Type 3 VWD is the most severe form of VWD and a new international study is underway to examine the use of prophylaxis.

The decreased survival of von Willebrand factor (VWF) in plasma has been implicated as a mechanism in a subset of type 1 von Willebrand disease (VWD) patients. We have previously reported that the ratio of plasma levels of VWF and its propeptide (VWFpp) can be used to identify patients with reduced VWF survival. In this study, we report the assay of VWFpp and VWF:Ag in 19 individuals recruited from 6 European centers within the MCMDM-1VWD study. Eight individuals had a VWF:Ag level less than 30 IU/dL. Seven of these patients had a robust desmopressin response and significantly reduced VWF half-life that was predicted by a markedly increased steady-state plasma VWFpp/VWF:Ag ratio. VWF mutations previously associated with reduced VWF survival were identified in each of the 7 individuals. Thus, a substantially increased ratio of steady-state VWFpp/VWF:Ag predicted a reduced VWF half-life in patients with markedly decreased VWF:Ag levels. These data indicate that a reduced VWF survival is found in a subpopulation of patients with type 1 VWD. The systematic assay of both plasma VWF and the VWF propeptide in moderately severe type 1 VWD patients may identify patients with a reduced VWF survival phenotype.

Background: Type 1 von Willebrand disease (VWD) is a congenital bleeding disorder characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF) in the absence of structural and/or functional VWF defects. Accurate assessment of the quantity and quality of plasma VWF is difficult but is a prerequisite for correct classification., Objective: To evaluate the proportion of misclassification of patients historically diagnosed with type 1 VWD using detailed analysis of the VWF multimer structure., Patients and Methods: Previously diagnosed type 1 VWD families and healthy controls were recruited by 12 expert centers in nine European countries. Phenotypic characterization comprised plasma VWF parameters and multimer analysis using low- and intermediate-resolution gels combined with an optimized visualization system. VWF genotyping was performed in all index cases (ICs)., Results: Abnormal multimers were present in 57 out of 150 ICs; however, only 29 out of these 57 (51%) had VWF ristocetin cofactor to antigen ratio below 0.7. In most cases multimer abnormalities were subtle, and only two cases had a significant loss of the largest multimers., Conclusions: Of the cases previously diagnosed as type 1 VWD, 38% showed abnormal multimers. Depending on the classification criteria used, 22 out of these 57 cases (15% of the total cohort) may be reclassified as type 2, emphasizing the requirement for multimer analysis compared with a mere ratio of VWF functional parameters and VWF:Ag. This is further supported by the finding that even slightly aberrant multimers are highly predictive for the presence of VWF mutations.

We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL or higher and partial response as VWF:RCo or FVIII:C lower than 50 IU/dL after infusion, but at least 3-fold the basal level. Complete response was observed in 83% of patients; partial in 13%; and no response in 4%. Patients with some abnormality of VWF multimeric pattern had significantly lower basal FVIII:C and VWF, lower VWF:RCo/Ag ratio, and less complete responses to desmopressin than patients with a normal multimeric pattern (P=.002). Patients with mutations at codons 1130 and 1205 in the D'-D3 domain had the greatest relative increase, but shortest FVIII and VWF half-lives after infusion. Most partial and nonresponsive patients had mutations in the A1-A3 domains. Response to desmopressin in these VWD patients seemed to be associated with the location of the causative mutation. The presence of subtle multimeric abnormalities did not hamper potential clinically useful responses, as in typical type 1 VWD.

Background and Objectives: A plasma-derived von Willebrand factor (VWF) concentrate with low factor VIII (FVIII) content was specifically developed to treat von Willebrand disease (VWD). Efficacy and safety were investigated by merging the results of two comparable protocols conducted prospectively in 5 European and 12 French centers., Methods and Results: Fifty patients with clinically severe VWD (72% had VWF ristocetin cofactor activity less than 10 IU dL(-1) and 46% had FVIII < 20 IU dL(-1)) were treated with the concentrate as the only therapy, except for clinical situations requiring a priming dose of FVIII to rapidly correct an intrinsic coagulation defect. A total of 139 spontaneous bleeding episodes were treated; only 53 (38%) needed a concomitant FVIII dose. Outcome was excellent or good in 89% of the episodes. Forty-four patients underwent 108 surgical or invasive procedures. Outcome was excellent or good in 95 scheduled procedures (only VWF was infused) and 13 emergency procedures (a priming FVIII dose was co-administered with the first VWF infusion). There were no thrombotic complications and none of the 18 patients with type 3 VWD developed anti-VWF or anti-FVIII antibodies., Conclusions: This concentrate safely and effectively provides hemostasis in patients with clinically severe VWD.

Two versions of conformation sensitive gel electrophoresis, fluorescent (F-CSGE) and manual (M-CSGE) techniques, were compared for mutation analysis of the von Willebrand factor gene. 56 PCRs were used to amplify all 52 exons of the gene in seven type 1 von Willebrand disease cases, plus a healthy control. One hundred and ninety-two samples were analyzed on each F-CSGE gel, compared with 40 on M-CSGE. 125 amplicons revealed bandshifts using F-CSGE, but only 101 by M-CSGE. Five mutations were detected by both techniques. F-CSGE detected 45 different polymorphisms whereas M-CSGE detected only 39. F-CSGE is high-throughput and more sensitive than M-CSGE.

Background: Presence of bleeding symptoms, inheritance and reduced von Willebrand factor (VWF) contribute to the diagnosis of type 1 von Willebrand disease (VWD). However, quantitative analysis of the importance of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) levels in the diagnosis is lacking., Objectives: To evaluate the relative contribution of VWF measurement to the diagnosis of VWD., Patients and Methods: From the MCMDM-1VWD study cohort, 204 subjects (considered as affected by VWD based on the enrolling Center diagnoses and the presence of linkage with the VWF locus) were compared with 1155 normal individuals. Sensitivity, specificity and diagnostic positive likelihood ratios (LR) of VWF:Ag and VWF:RCo were computed., Results: ABO blood group was the variable most influencing VWF levels, but adjustment of the lower reference limit for the ABO group did not improve sensitivity and specificity of VWF:Ag or VWF:RCo. The lower reference limit (2.5th percentile) was 47 IU dL(-1) for both VWF:Ag and VWF:RCo and showed similar diagnostic performance [receiver-operator curve area: 0.962 and 0.961 for VWF:Ag and VWF:RCo, respectively; P = 0.81]. The probability of VWD was markedly increased only for values below 40 IU dL(-1) (positive LR: 95.1 for VWF:Ag), whereas intermediate values (40 to 60 IU dL(-1)) of VWF only marginally indicated the probability of VWD., Conclusions: Although the conventional 2.5 lower percentile has good sensitivity and specificity, only VWF:Ag or VWF:RCo values below 40 IU dL(-1) appear to significantly indicate the likelihood of type 1 VWD. The LR profile of VWF level could be used in a diagnostic algorithm.

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%.

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factorVIII (FVIII) and is caused by mutations in the D' or D3 domain of mature VWF. We now report a French patient with an atypical 2N VWD phenotype associating FVIII deficiency with plasmaVWF unable to bind FVIII (undetectableVWF:FVIIIB) but with an abnormal multimeric profile. This patient is heterozygous for both the frequent R854Q type 2NVWD mutation and a novel R763G mutation at the cleavage site between VWF propeptide and mature VWF. Four children of the patient displayed moderately decreased VWF:FVIIIB of plasma VWF and were heterozygous for either the R763G or the R854Q mutation. Children with the R763G mutation displayed the same abnormal multimeric profile as their father. Recombinant VWF (rVWF) expression studies performed in COS-7 cells showed that the R763G mutation subtly affects its multimeric profile and dramatically impairs its FVIII binding function. Furthermore, the characteristics of hybrid G763/Q854 rVWF resulting from cotransfection experiments were in agreement with the type 2N VWD diagnosis of the patient. We conclude that R763G is a new type 2N VWD mutation located in the VWF propeptide which alters the proteolytic processing of VWF and consequently its binding to FVIII.

Type 2B von Willebrand disease (VWD) is characterised by an increased affinity of von Willebrand factor (VWF) for its platelet receptor glycoprotein Ib (GPIb). This feature is usually studied in vitro by a ristocetin-dependent VWF platelet-binding assay, which has some limitations as it requires [e.g. (radio)-labelled anti-VWF antibodies and normal formaldehyde-fixed platelets]. We, here, extended the applicability of an enzyme-linked immunosorbent assay-based method previously described for the measurement of ristocetin co-factor activity that used a recombinant fragment of GPIb (rfGPIb alpha) and horseradish peroxidase-labelled rabbit anti-human VWF antibodies for measuring the captured ristocetin-VWF complexes on the rfGPIb alpha. Thirty-one type 2B VWD patients from 15 families with eight different known mutations were studied. VWF in plasma from 28 of these patients bound better than normal VWF at 0.2 mg/ml ristocetin, with the ratio, optical density (OD) patient/OD normal pool plasma, higher than 1.8. For two of the three other patients with no enhanced response of plasma VWF, the platelet lysate VWF showed an enhanced binding capacity; for the last patient, the results in other members of the family are unequivocal. We conclude that, this new method for measurement of plasma or platelet VWF-binding capacity offers great advantages for correct type 2B VWD diagnosis.

Background: von Willebrand disease (VWD) type 1 is a congenital bleeding disorder caused by genetic defects in the von Willebrand factor (VWF) gene and characterized by a reduction of structurally normal VWF. The diagnosis of type 1 VWD is difficult because of clinical and laboratory variability. Furthermore, inconsistency of linkage between type 1 VWD and the VWF locus has been reported., Objectives: To estimate the proportion of type 1 VWD that is linked to the VWF gene., Patients and Methods: Type 1 VWD families and healthy control individuals were recruited. An extensive questionnaire on bleeding symptoms was completed and phenotypic tests were performed. Linkage between VWF gene haplotypes and the diagnosis of type 1 VWD, the plasma levels of VWF and the severity of bleeding symptoms was analyzed., Results: Segregation analysis in 143 families diagnosed with type 1 VWD fitted a model of autosomal dominant inheritance. Linkage analysis under heterogeneity resulted in a summed lod score of 23.2 with an estimated proportion of linkage of 0.70. After exclusion of families with abnormal multimer patterns the linkage proportion was 0.46. LOD scores and linkage proportions were higher in families with more severe phenotypes and with phenotypes suggestive of qualitative VWF defects. About 40% of the total variation of VWF antigen could be attributed to the VWF gene., Conclusions: We conclude that the diagnosis of type 1 VWD is linked to the VWF gene in about 70% of families, however after exclusion of qualitative defects this is about 50%.

Background: A quantitative description of bleeding symptoms in type 1 von Willebrand disease (VWD) has never been reported., Objectives: The aim was to quantitatively evaluate the severity of bleeding symptoms in type 1 VWD and its correlation with clinical and laboratory features., Patients and Methods: Bleeding symptoms were retrospectively recorded in a European cohort of VWD type 1 families, and for each subject a quantitative bleeding score (BS) was obtained together with phenotypic tests., Results: A total of 712 subjects belonging to 144 families and 195 controls were available for analysis. The BS was higher in index cases than in affected family members (BS 9 vs. 5, P < 0.0001) and in unaffected family members than in controls (BS 0 vs. -1, P < 0.0001). There was no effect of ABO blood group. BS showed a strong significant inverse relation with either von Willebrand ristocetin cofactor (VWF:RCo), von Willebrand antigen (VWF:Ag) or factor VIII procoagulant activity (FVIII:C) measured at time of enrollment, even after adjustment for age, sex and blood group (P < 0.001 for all the four upper quintiles of BS vs. the first quintile, for either VWF:RCo, VWF:Ag or FVIII:C). Higher BS was related with increasing likelihood of VWD, and a mucocutaneous BS (computed from spontaneous, mucocutaneous symptoms) was strongly associated with bleeding after surgery or tooth extraction., Conclusions: Quantitative analysis of bleeding symptoms is potentially useful for a more accurate diagnosis of type 1 VWD and to develop guidelines for its optimal treatment.

Objective: In order to correct the primary von Willebrand factor (VWF) defect and avoid supra-physiologic plasma levels of factor VIII, a pure VWF concentrate almost devoid of FVIII was developed and used in France since 1989., Methods: The pharmacokinetic (PK) profile of the most recent version of this concentrate (Wilfactin; LFB, Les Ulis, France), treated with three virus-inactivation/removal methods (solvent/detergent, 35 nm filtration, dry heat treatment), was investigated in 25 patients. Seventeen patients with various types of clinically severe von Willebrand disease (VWD) were included in a crossover, randomized trial carried out in five European centers and comparing Wilfactin with concentrates containing both FVIII and VWF (FVIII/VWF). Eight type 3 VWD patients were included in another trial carried out in six French centers comparing Wilfactin with its previous version (Facteur Willebrand-LFB; LFB) that adopted one virus-inactivation method only., Results: For both the measurements evaluated in this study (VWF antigen, VWF:Ag; and VWF ristocetin co-factor activity, VWF:RCo), Wilfactin had a PK profile similar to that of the FVIII/VWF concentrates and of Facteur Willebrand-LFB. VWF:RCo and VWF:Ag recoveries were 2.1 +/- 0.3 and 1.8 +/- 0.3 per IU kg(-1), respectively, and the half-lives were 12.4 +/- 1.8 and 15.9 +/- 1.5 h. The FVIII synthesis rate was 5.8 +/- 1.0 IU dL(-1) h(-1), with a half-life of 15.8 +/- 2.4 h., Conclusion: The PK of VWF and FVIII have not been altered by the three virus-inactivation/removal steps during the manufacturing of Wilfactin.

This study prospectively evaluated the rate of biologic response to desmopressin (DDAVP) in 66 patients with type 1 or 2 von Willebrand disease (VWD), each of whom had, on the basis of available records, a clinically significant bleeding history and at least one of the following laboratory abnormalities: bleeding time (BT) longer than 15 minutes, ristocetin cofactor activity (VWF:RCo) less than 10 IU/dL, factor VIII coagulant activity (FVIII:C) less than 20 IU/dL (severe VWD). Before the study, responsive patients were defined as those who, 2 hours after infusion of 0.3 microg/kg DDAVP, had increased baseline values of VWF:RCo and FVIII:C by at least 3-fold and achieved levels of at least 30 IU/dL for both and a BT of 12 minutes or less. The rate of biologic response varied according to VWD types and was higher in type 1 (7 of 26, 27%) than in type 2 (7 of 40, 18%) (type 2A [1 of 15, 7%], type 2M [3 of 21, 14%], type 2N [3 of 4, 75%]). Mutations in the VWF gene were previously known or newly identified in most patients with types 2A (n = 15 of 15), 2M (n = 15 of 21), and 2N (n = 4 of 4), but in none of those with type 1 VWD. Genotype provided more information than phenotype in predicting individual responses to DDAVP only in patients with 2A and 2N VWD. This prospective study showed that the rate of biologic response to DDAVP is relatively low not only in type 2 but also in type 1 VWD when uniform and stringent criteria for patient selection and responsiveness are applied.

Background: Aortic-valve stenosis can be complicated by bleeding that is associated with acquired type 2A von Willebrand syndrome. However, the prevalence and cause of the hemostatic abnormality in aortic stenosis are unknown., Methods: We enrolled 50 consecutive patients with aortic stenosis, who completed a standardized screening questionnaire to detect a history of bleeding. Forty-two patients with severe aortic stenosis underwent valve replacement. Platelet function under conditions of high shear stress, von Willebrand factor collagen-binding activity and antigen levels, and the multimeric structure of von Willebrand factor were assessed at base line and one day, seven days, and six months postoperatively., Results: Skin or mucosal bleeding occurred in 21 percent of the patients with severe aortic stenosis. Platelet-function abnormalities under conditions of high shear stress, decreased von Willebrand factor collagen-binding activity and the loss of the largest multimers, or a combination of these was present in 67 to 92 percent of patients with severe aortic stenosis and correlated significantly with the severity of valve stenosis. Primary hemostatic abnormalities were completely corrected on the first day after surgery but tended to recur at six months, especially when there was a mismatch between patient and prosthesis (with an effective orifice area of less than 0.8 cm2 per square meter of body-surface area)., Conclusions: Type 2A von Willebrand syndrome is common in patients with severe aortic stenosis. Von Willebrand factor abnormalities are directly related to the severity of aortic stenosis and are improved by valve replacement in the absence of mismatch between patient and prosthesis., (Copyright 2003 Massachusetts Medical Society)

The pathophysiology of acquired von Willebrand syndrome (AVWS), a rare bleeding disorder, is not fully understood. Circulating antibodies to Von Willebrand factor (VWF) are found in patients with AVWS associated with lymphoproliferative disorders but these autoantibodies are difficult to detect with routine laboratory tests and neutralisation assays. We have developed a simple enzyme-linked immunosorbent assay (ELISA) to detect serum antibody binding to VWF protein immobilized on polystyrene plates. Ten patients with AVWS were studied, eight of whom also had lymphoproliferative disorders. We found antibodies in eight patients; all of them were positive for IgG and five were also positive for IgM. This simple method appears to be more sensitive than functional assays, which failed to identify two of the patients who were positive with the ELISA. In conjunction with other tests, this ELISA method may be useful for demonstrating the immunological mechanism underlying some cases of AVWS. Such patients would qualify for intravenous immunoglobulin therapy, which can correct the clotting disorder.

In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D' domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site-directed mutagenesis and transient expression in COS-7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

In this study, we reinvestigated a 20-year-old woman, the first cousin of two brothers with severe haemophilia A. This patient was previously assumed to be a carrier of haemophilia A due to her FVIII deficiency. We identified a novel FVIII gene mutation in the family and demonstrated that the FVIII deficiency in this female patient did not result from this gene mutation, but was linked to molecular defects in the von Willebrand factor gene.

The present diagnostic assay for type 2N von Willebrand disease (VWD) is based on the in vitro measurement of the capacity of plasma von Willebrand factor (VWF) to bind exogeneous factor VIII (VWF:FVIIIB). We report a method using only commercially available reagents that is easy to perform. This method has been validated in a cohort of 144 patients with FVIII/VWF ratios < 0.6 using a plasma control mixture representative of intermediate VWF:FVIIIB. In total, 15 patients were diagnosed with markedly decreased VWF:FVIIIB and five patients were shown to have moderately decreased VWF:FVIIIB. Specific type 2N mutations were identified in all these patients.

Type 2N von Willebrand disease encompasses all patients with factor VIII deficiency caused by a markedly decreased affinity of von Willebrand factor for factor VIII. It is recessively inherited and clinically similar to mild haemophilia. The differential biological diagnosis is of major importance for providing the optimal treatment and relevant genetic counselling. This accurate diagnosis is based on an evaluation of the factor VIII-binding capacity of plasma von Willebrand factor. Furthermore, molecular biology techniques allow the identification of missense mutations in the von Willebrand factor gene. All of these induce the substitution of amino acid residues located in the N terminal part of the mature von Willebrand factor molecule, which contains the factor VIII binding site. Most of them induce a classical type 2N von Willebrand disease phenotype with factor VIII deficiency but a normal level and multimeric pattern of von Willebrand factor.

The study identified 10 patients from 6 families with prolonged bleeding time, decreased von Willebrand factor (vWF) ristocetin cofactor activity (RCoF) to vWF:Ag (antigen) ratio, and reduced ristocetin-induced platelet agglutination as well as ristocetin- or botrocetin-induced binding of plasma vWF to platelet glycoprotein Ib (GpIb). In addition, all patients showed a decrease of intermediate-molecular-weight (intermediate-MW) and high-molecular-weight (HMW) multimers of vWF. In the heterozygous state, a cysteine-to-threonine (C --> T) transversion was detected at nucleotide 4193 of the VWF gene of all patients and lead to the arginine (R)522C substitution in the A1 loop of vWF mature subunit (R1315C in the preprovWF). By in vitro mutagenesis of full-length complementary DNA (cDNA) of vWF and transient expression in COS-7 cells, the mutated C552 recombinant vWF (C552rvWF) was found to exhibit decreased expression, abnormal folding, and lack of intermediate-MW and HMW multimers. In addition, direct binding of botrocetin to C552rvWF, as well as ristocetin- and botrocetin-induced binding of C552rvWF to GpIb, was markedly decreased. Although being localized in an area of the A1 loop of vWF where most of the type 2B mutations that induce a gain-of-function have been identified, the R552C mutation induces a 2A-like phenotype with a decrease of intermediate-MW and HMW multimers as well as a loss-of-function of vWF in the presence of either ristocetin or botrocetin. (Blood. 2001;97:952-959)

We report 2 new mutations identified in 3 patients and characterized by the markedly decreased affinity of von Willebrand factor (vWF) for factor VIII (FVIII). Patients 2 and 3, who have a typical type 2N phenotype, were found to be compound heterozygous for Arg91Gln and Cys25Tyr or Cys95Phe, respectively. Patient 1, who is the first cousin of patient 2, had an FVIII binding defect of vWF, low levels of vWF, and multimeric impairment. She was found to be compound heterozygous for the mutations Cys25Tyr and a stop codon (D93ter) in exon 4. Transient expression of recombinant vWF (rvWF) containing either Cys25Tyr or Cys95Phe mutations resulted in mutated rvWF with markedly reduced FVIII binding ability, multimeric structure impairment, and a significant decrease in the vWF expression level. Moreover, the use of anti-vWF monoclonal antibodies that inhibit the FVIII binding showed that these 2 mutations likely induce a conformational change in the D' domain. These results show that the native conformation of the D' domain of vWF is not only required for FVIII binding but also for normal multimerization and optimal secretion.

In type 2N von Willebrand disease (vWD), von Willebrand factor (vWF) is characterized by normal multimeric pattern, normal platelet-dependent function, but a markedly decreased affinity for factor VIII (FVIII). In this report, we describe the case of a vWD patient who has an abnormal vWF multimers distribution associated with a markedly decreased vWF ability to bind FVIII. Sequencing analysis of patient's vWF gene showed, at heterozygous state, a G-->A transition resulting in the substitution of Asn for Asp at position 116 of the mature vWF subunit and a C-->T transition, changing the codon for Arg 896 into a stop codon. His sister who has a subnormal vWF level, but a normal FVIII/vWF interaction, was found to be heterozygous for the Arg896ter mutation only. Recombinant vWF (rvWF) containing the candidate (Asn116) missense mutation was expressed in COS-7 cells. The expression level of Asn116rvWF was significantly decreased compared with wild-type rvWF. The multimeric pattern of Asn116rvWF was greatly impaired as shown by the decrease in high molecular weight forms. The FVIII binding ability of Asn116rvWF was dramatically decreased. These data show that the Asp116Asn substitution is the cause of both the defective FVIII/vWF interaction and the impaired multimeric pattern observed in the patient's vWF. The monoclonal antibody 31H3 against D' domain of vWF (epitope aa 66-76) that partially inhibits the FVIII binding and recognizes only nonreduced vWF, showed a decreased ability to bind Asn116rvWF when used as capture-antibody in enzyme-linked immunosorbent assay (ELISA). This result suggests that a potential conformation change in the D' domain is induced by the Asp116Asn substitution, which is localized in the D3 domain.

Part of the French clinical experience using the solvent/detergent (S/D) treated VHP von Willebrand factor (vWF) concentrate (LFB, Les Ulis, France) characterized by a high vWF:RCoF specific activity and a low factor VIII (FVIII) content is reported. Since 1989 this concentrate has been routinely used in clinical practice taking into account the vWF:RCoF given by the manufacturer. Seventy-five patients with von Willebrand disease (type 1: 42, 2A: 11, 2B: 5, 2N: 6, 3: 4, acquired: 7) were treated on 99 occasions either to control spontaneous bleedings (15) or to prevent haemorrhagic risks associated with minor (< 5 days of treatment) (48) or major (5 days of treatment) (36) surgery including seven knee or hip-replacements. Forty lots of concentrate were used containing 58 +/- 13 U mL-1 vWF:RCoF with less than 10 units of FVIII per 100 units vWF:RCoF. Patients with type 2N were analysed separately. With the exception of gastro-intestinal bleedings, spontaneous bleedings were generally stopped after few infusions of 40-47 U kg-1 vWF:RCoF. Patients having more than 20 U dL-1 FVIII were treated on 54 surgical occasions with one preoperative infusion (51-55 U kg-1 vWF:RCoF) which allowed an increase in FVIII concentration to a mean level of 67-88 U dL-1. Patients with less than 20 U dL-1 were either treated with two preoperative infusions of vWF, 12 or 24 h apart (11 cases) or received a FVIII injection immediately after the preoperative infusion of vWF (10 cases). During the postoperative period vWF alone (30-35 U kg-1 vWF:RCoF) allowed FVIII to be kept at a mean level of 118-138 U dL-1 not exceeding 180 U dL-1. Patients with type 2N were treated taking in account only their baseline FVIII concentration. No haemorrhagic complications occurred in any of the patients. Thus it was found to be feasible and practical to manage replacement therapy in patients with von Willebrand disease (whatever the type or the circumstances) on the basis of the vWF:RCoF activity of the concentrate.

Normal endothelium, as well as megakaryocytes, produces and secretes von Willebrand factor which plays a pivotal role in haemostasis. It mediates platelet adhesion and shear-stress-induced aggregation at the site of vessel wall injury and also serves as a carrier protein of factor VIII, an essential cofactor in coagulation. The quantitative or qualitative abnormalities of von Willebrand factor lead to von Willebrand disease, the most common congenital bleeding disorder. Over the past few years major progress in the knowledge of the synthesis, structure and functions of von Willebrand factor have led to improve the diagnosis and treatment of the different molecular forms of von Willebrand disease.

Willebrand's disease, the most frequent inborn coagulopathy, is defined as a deficiency in Willebrand's factor required for normal hemostasis as a mediator in platelet adhesion to the subendothelium and which also contributes to plasma coagulation pathway (by preserving the coagulating activity of factor VIII). Classically, Willebrand's disease improves somewhat during pregnancy. We followed 15 pregnancies in 12 patients with Willebrand's disease in an attempt to determine the best management strategy to reduce the risk of bleeding during delivery. This risk can be assessed on the basis of prior history of bleeding and the laboratory tests and reduced by administration of concentrated Willebrand's factor at 8 months gestation. In patients with type I disease, desmopressin is proposed as curative treatment during the post partum period. Antenatal diagnosis is possible in the most severe forms. Programmed delivery is recommended. Spinal analgesia is contraindicated.

We evaluated the effectiveness of desmopressin to control bleeding of patients with coagulation defects during dental surgery. Thirty-five patients, mainly with moderate and mild hemophilia and Willebrand disease, were undergoing dental extractions (over 80 extractions in total). Bleeding was successfully prevented in 28 patients with the use of a combined treatment incorporating IV desmopressin, an antifibrinolytic agent (tranexamic acid), and local methods (surgical glue and compression techniques). Seven patients had a bleeding episode after dental extraction, which was controlled in two cases by repeated injection of desmopressin and in another two by local methods; Factor VIII substitutive treatment was needed in only three patients. Desmopressin offers an alternative to blood products to control bleeding risk in patients with moderate and mild coagulation defects. Our experience tends to specify the mode of administration of both desmopressin and the associated treatments. Our findings suggest that desmopressin can be used in conjunction with other treatments to prevent bleeding in patients with coagulation defects who undergo dental surgery. This work highlights the concept of multifactorial medical care of these patients in which desmopressin plays a major role.

A case of acquired von Willebrand's disease (AvWD) associated with an adrenal cortical carcinoma is reported. The circulating highest molecular weight multimers (HMWM) of von Willebrand factor (vWF) were decreased when assessed by SDS-agarose plasma electrophoresis, leading to the diagnosis of type II AvWD. No forms of inhibitor could be detected in the plasma of the patient. In contrast, indirect immunoperoxidase studies with a monoclonal antibody to vWF demonstrated an absorption of vWF into malignant cells. Infusion of a vWF-FVIII concentrate, containing significant amounts of HMWM of vWF, allowed surgical resection of the tumour. After the first infusion of the concentrate, the vWF-RCo recovery was found to be low (38%) compared to the vWF:Ag (75%) and FVIII:C (163%) recoveries. The resolution of all biological signs of vWD, including the abnormal multimeric pattern, in the post-operative period was prompt and permanent. Therefore, the absorption of the HMWM of vWF by carcinomatous cells appears to represent a likely pathophysiological mechanism responsible for the AvWD syndrome in this patient.

This study was carried out to assess the clinical efficacy in von Willebrand's disease (vWD) of a new, very high purity (VHP), solvent/detergent (SD)-treated, vWF concentrate (VHP Human von Willebrand Factor Concentrate, Biotransfusion) characterized by a high specific ristocetin cofactor (vWF:RCo) activity and a low factor VIII (FVIII) coagulant activity (FVIII:C). Nine patients (four type I, one type IIA, one type IIB, one type IIC, one type III and one acquired type II) were infused on 13 occasions including a pharmacokinetic study. Satisfactory haemostasis was achieved in all cases, including the treatment of spontaneous haemorrhages and the prevention of bleeding following surgery. The bleeding time was corrected for 6-12 h in 6/9 patients and shortened in the others. Furthermore, it was shown that the plasma vWF multimeric pattern of types II and III patients was greatly improved. When measured in eight patients 1 h after infusion, the vWF:RCo recovery was 77.3 (+/- 10.7)% while the F VIII:C recovery was strikingly higher (876 +/- 906%). This high recovery is likely related to the predominant 'pseudo-synthesis' of FVIII following the restoration of normal vWF levels. Maximum levels of FVIII:C occurred 6-12 h after the first infusion and normal levels of FVIII:C were maintained throughout the treatments with a dosage of 26-39 IU/kg vWF:RCo and only 0.2-5 IU/kg FVIII:C. The half-lives of the vWF-related parameters determined in a type III vWD patient were 20.6 h for vWF antigen, 17.8 h for vWF:RCo, 14 h for the high molecular weight multimers of vWF, 55.3 h for FVIII:Ag and 74 h for FVIII:C. In conclusion, it does not appear necessary that vWF concentrates intended for the treatment of vWD should contain FVIII in addition to vWF to be clinically effective in most patients.

Twenty-six members of four generations of one family in which a man was diagnosed in 1961 as having von Willebrand's disease (vWD) have been studied. Subtype IIB vWD and autosomal dominant inheritance was identified in 19 individuals with bleeding signs varying in severity and frequency. The absence of high molecular weight multimers of plasma von Willebrand factor (vWf) and the heightened interaction of plasma vWf with platelets in the presence of low ristocetin concentrations were consistent in all affected subjects. Nevertheless the degree of these abnormalities was variable without clearcut linkage to the severity of clinical symptoms. Thrombocytopenia was present only in the adult affected family members and the platelet count appeared to be age-dependent. The investigation of this family provides further evidence of the phenotypic variability in the vWf-platelet interactions within this vWD subtype.

An acquired von Willebrand's disease developed in two patients in association with a monoclonal gammopathy plus a Sjögren's syndrome and a chronic lymphocytic leukaemia (CLL). In both cases a plasma inhibitor to von Willebrand factor (vWf) was suspected and characterized after plasma gel filtration. The inhibitor was shown to be entirely complexed with vWf and was only demonstrated after complex dissociation by heating. The inhibitor was able to inhibit the binding of 125I-vWf to platelets in the presence of ristocetin in both cases and to thrombin-stimulated platelets in one case. In the two patients, the highest molecular weight multimers (HMWM) of vWf were absent when assessed by sodium dodecyl-sulphate agarose plasma electrophoresis. Intravenous infusion of 1-deamino-(8-D-arginine) vasopressin (DDAVP) resulted in the appearance of the HMWM in both cases and of the satellite bands of each multimer subunit which were lacking prior to the infusion in one patient. After transfusion of a VIII/vWf concentrate containing a significant amount of HMWM, there was a rapid plasma clearance of the vWf-related activities and of the HMWM when compared to that seen in a patient with type III constitutional vWD. We conclude that in the two patients studied the coagulation defect was related to the presence of a circulating inhibitor to vWf which could be responsible for the disappearance of the HMWM from plasma.

The frequency of HLA antigens was determined in 28 unrelated patients with Willebrand's disease and in 326 healthy controls. There was no significant difference between the two groups for any of the HLA antigens tested. Segregation analysis processed among the families of 26 of these patients didn't allow us to find any disorder of the HLA antigens distribution in the patient's group: transmission of the disease and transmission of the HLA haplotypes appear to be independant in 25 of 26 families studied. There is no linkage and no association between the Willebrand's disease genes and the HLA system genes.