Corrigendum to “A family having type 2B von Willebrand disease with an R1306W mutation: Severe thrombocytopenia leads to the normalization of high molecular weight multimers” [Thrombosis Research 125 (2010) e17–e22]

Abstract Abstract 543 Background: Type 2 A and 2 M von Willebrand Disease (VWD) are both characterized by the presence of a dysfunctional von Willebrand factor (VWF) and a variable bleeding tendency. The bleeding incidence and possible clinical differences between these two types have never been investigated prospectively in a large number of patients. Aims and design of the study: To investigate clinical history and determinants of bleeding in a cohort of patients (pts) with type 2 A and M VWD characterized by mutations over 24 months of follow-up. Patients and Methods: 10 families with type 2 A VWD (47 patients) and 15 with type 2 M (58 patients) with type 2 M VWD were diagnosed according to the recommendations of the ISTH-SSC subcommittee on VWF and prospectively followed-up from April 2007 to March 2009. Bleeding score (BS) was calculated in all the patients at enrollment by the administration of the same questionnaire tested in type 1 VWD. In case of VWF:RCo levels < 10 U/dL, all patients were tested with a more sensitive ELISA assay to calculate ratios between VWF:RCo and VWF:Ag. VWF mutations were identified in all the patients. Results: The 105 patients were characterized by the following mutations: type 2 A R202W/R1583Q 1 pt, S1506L 11 pts, S1543F 1 pt, R1596W 3 pts, V1607D 8 pts, I1628T 1 pt, G1629R 1 pt, G1631D 9 pts, V1665E 8 pts, Q2520P 2 pts, multiple changes 1 pt. Type 2 M: L1278P 6 pts, R1315L 3 pts, R1315C 9 pts, Y1321C 9 pts, L1361W 4 pts, R1374H 23 pts, C1927/c.8155+6C>T 1 pt, c.3831del-3 3 pts. The table summarizes the main demographic and laboratory characteristics. All the patients had a VWF:RCo/VWF:Ag ratio <0.6. The mean BS and VWF:Ag were significantly higher in type 2 A (P < 0.01). No correlation between VWF:RCo levels and the severity of BS was observed. During follow-up, the bleeding episodes in patients with type 2 M and R1374H or R1315C mutations were very few and mild while they were recurrent and severe in those with type 2 A and V1665E or G1631D mutations, the only laboratory difference being the lack of high molecular weight multimers in type 2 A VWD. Furthermore, 44 gastrointestinal bleeding episodes occurred in 15 patients with type 2 A (range 1–7) compared to 8 episodes in 2 patients with type 2 M (range 2–6). Older age was strongly related to the risk of recurrence of this type of bleeding. Conclusions: Bleeding tendency in type 2 A VWD is higher than that of type 2 M VWD and it is not related to the reduction of FVIII:C and VWF activity. The risk of gastrointestinal bleeding is greater in type 2 A and might be related to the lack of high molecular weight multimers and older age. Further analysis are required to evaluate the definite impact of some mutations on the different bleeding tendency. Disclosures: No relevant conflicts of interest to declare.

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Von Willebrand Factor Collagen Binding Provides a Sensitive Screen for Identification of Type 2A and 2B Von Willebrand Disease

Blood ◽

10.1182/blood.v118.21.379.379 ◽

2011 ◽

Vol 118 (21) ◽

pp. 379-379

Author(s):

Veronica H Flood ◽

Joan Cox Gill ◽

Kenneth D Friedman ◽

Pamela A Christopherson ◽

Paula M. Jacobi ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Von Willebrand Disease ◽

Collagen Binding ◽

The Mean ◽

Von Willebrand ◽

Willebrand Factor ◽

Normal Controls ◽

High Molecular Weight Multimers

Abstract Abstract 379FN2 Collagen binding is an easily performed test of von Willebrand factor (VWF) function but its role in clinical evaluation is still debated. Analysis of multimer distribution, on the other hand, is time-consuming and technically challenging. We hypothesized that VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and collagen binding (VWF:CB) could identify the subset of von Willebrand disease (VWD) cases in which multimer analysis would be informative. Subjects from the Zimmerman Program for the Molecular and Clinical Biology of VWD were analyzed for VWF:Ag, VWF:RCo, VWF:CB (with type III human placental collagen), multimer distribution, and full VWF exon sequencing. Normal controls as well as patients with type 1, 2A, 2B, 2M, and 2N VWD were analyzed. The mean VWF:CB/VWF:Ag ratio for subjects with normal multimers was 1.10, while the mean ratio for subjects with abnormal multimers was 0.51 (p<0.001). When results were restricted to those subjects with confirmed type 2A or type 2B mutations, however, the mean ratio for subjects with abnormal multimers decreased to 0.41 (p<0.001 compared to those with normal multimers). For the 146 normal controls with multimer results available, 2 had absence of the highest molecular weight multimers, but normal collagen binding, normal bleeding scores, and no evidence of a VWF gene mutation, suggesting that the multimer results represented assay artifact. 354 type 1 subjects were examined; of those, 12 had abnormal multimer patterns. 7 had loss of the high molecular weight multimers. Of these, 5 had known type 1 VWD mutations and normal VWF:CB/VWF:Ag ratios, possibly representing sample artifacts rather than a true multimer abnormality, as no multimer issues have been previously reported for these mutations. One had no mutation found and one had a type 2A mutation. 2 had a full spectrum of multimers with relatively increased staining of the lower molecular weight bands; both with novel A1 domain mutations that are currently under investigation. 3 had larger than normal multimers observed, all with normal VWF:CB/VWF:Ag ratios. Of the 342 type 1 subjects with normal multimers, only one had a VWF:CB/VWF:Ag ratio of <0.7, likely due to very low values (VWF:CB of 2 and VWF:Ag of 4). There were 36 type 2A subjects available for analysis. 27 had loss of high molecular weight multimers. Only 3 of those had VWF:CB/VWF:Ag ratios >0.7, but none of those subjects had VWF mutations consistent with type 2A VWD. 7 subjects had a shift from high to low molecular weight multimers, 4 with VWF:CB/VWF:Ag ratios >0.7 and either known type 1 mutations or novel VWF gene mutations. 2 subjects had normal multimer distribution, one with a type1 VWD mutation and one with a novel mutation. Characterization of these novel mutations is in progress. All the 17 type 2B subjects had loss of high molecular weight multimers and abnormal collagen binding, with a VWF:CB/VWF:Ag ratio <0.7. Interestingly, however, not all had a reduced VWF:RCo/VWF:Ag ratio, suggesting VWF:CB would be required in addition to VWF:RCo if multimer distribution was omitted in initial evaluation of this type of VWD. Of 18 type 2M subjects, only one had an abnormal multimer distribution. That subject had no mutations in the VWF coding sequence and normal VWF:CB, although the VWF:RCo/VWF:Ag ratio was low at 0.53. Repeat analysis of a new sample from this subject is pending. All 7 type 2N VWD subjects had normal multimers and VWF:CB/VWF:Ag ratios >0.7. In our population, with the exception of mutations that are yet to be characterized, the combination of VWF:Ag, VWF:RCo and VWF:CB was sufficient to categorize patients as normal, type 1, type 2A, 2B or 2M in the before multimer analysis. These findings suggest that VWF:CB is a sensitive screen for detection of an abnormal multimer distribution. Collagen binding is technically much easier to perform, allowing multimer analysis to be reserved for those cases with low VWF:RCo/VWF:Ag or low VWF:CB/VWF:Ag ratios. Disclosures: No relevant conflicts of interest to declare.

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Laboratory Predictors Of Loss Of High Molecular Weight Multimers In Patients With Cardiovascular Disease-Associated Acquired Von Willebrand Syndrom

Blood ◽

10.1182/blood.v122.21.335.335 ◽

2013 ◽

Vol 122 (21) ◽

pp. 335-335

Author(s):

Ewa M. Wysokinska ◽

Dong Chen ◽

Joseph L Blackshear

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Von Willebrand Factor ◽

Bleeding Risk ◽

Protein Electrophoresis ◽

Von Willebrand Disease ◽

Cardiovascular Disorders ◽

Von Willebrand ◽

Willebrand Factor ◽

High Molecular Weight Multimers

Abstract Background Association of acquired von Willebrand syndrome (AVWS) with various cardiovascular (CV) disorders such as cardiac valve disease and hypertrophic cardiomyopathy (HCM) is well known and documented. The mechanism is thought to be related to shear stress induced loss of high molecular weight multimers (HMWM). The gold standard test to assess for loss of HMWM is von Willebrand protein electrophoresis and then visual assessment of loss of high molecular weight bands. This is both a costly and subjective test. Ratio of von Willebrand factor activity to antigen level is useful in patients with type IIA Von Willebrand Disease caused by loss of HMW multimer, but its sensitivity to detect CV-associated AVWS is unknown. Aim Our aim was to test whether routine VWF laboratory tests could be used to predict which patients with CV conditions are going to have high molecular weight multimer loss. We also aimed to assess whether these tests could be used to predict bleeding risk in patients with CV disorders. Methods We prospectively collected laboratory data of 234 patients with cardiovascular disorders known to be associated with AVWS: aortic stenosis (66), aortic insufficiency(22), aortic and mitral valve prostheses(38), mitral valve regurgitation (51) and hypertrophic cardiomyopathy(57). All patients had Von Willebrand factor antigen (VWF:Ag), Von Willebrand factor activity by latex method (VWF:Ltx), platelet function testing via PFA-100 CADP as well as von Willebrand factor multimers tested. All patients also completed a bleeding questionnaire. We used logistic regression model to calculate the relationship between the VWF:Ltx/VWF:Ag ratio and loss of high molecular weight multimers. Same analysis was performed for PFA-100. We also tested these associations for bleeding risk. Results Mean value for VWF:Ag was 142 IU/dL, VWF:Ltx 121%, PFA-100 151 seconds and 0.86 for the VWF:Ltx/Ag ratio. Over a half of patients (56%) had VWF multimer loss noted on protein electrophoresis testing and a quarter reported bleeding on bleeding questionnaire. The ratio of VWF:Ltx to VWF:Ag had strong correlation with HMW multimer loss (p<0.001) with AUC of 0.77. Correlation with PFA-100 was even stronger with AUC of 0.83. The ratio cut off value of 0.83 had sensitivity of 60% and specificity of 83% in predicting multimer loss. With the cut off of 0.77, specificity reached 95%. With PFA 100 value of 118 seconds, specificity was 76% and sensitivity was 80%. Increasing the cut off to 198 seconds improved the specificity to 95%. The association with bleeding was present for PFA-100 (p=0.01), but did not exist for the Ltx/Ag ratio. Conclusions PFA-100 CADP as well as VWF:Ag and VWF:Ltx can be used to detect acquired Von Willebrand disease in patients with cardiovascular disorders and may decrease the need for costly and time consuming testing of multimers. PFA-100 CADP also correlates with the bleeding risk in these patients. Disclosures: No relevant conflicts of interest to declare.

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A Modified Model of Type 2B von Willebrand Disease: Taking ADAMTS13-Mediated Cleavage out of the Equation

Blood ◽

10.1182/blood.v116.21.23.23 ◽

2010 ◽

Vol 116 (21) ◽

pp. 23-23

Author(s):

Mia Golder ◽

Cynthia M. Pruss ◽

Kate Sponagle ◽

Carol Hegadorn ◽

Erin Burnett ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Binding Affinity ◽

Full Range ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Platelet Counts ◽

Platelet Binding ◽

Von Willebrand ◽

High Molecular Weight Multimers

Abstract Abstract 23 Introduction: Type 2B von Willebrand disease (2B VWD), a qualitative variant of VWD, is characterized by thrombocytopenia and loss of high molecular weight multimers. 2B VWF has both an increased affinity for platelet glycoprotein Ibα (GPIbα) and increased susceptibility to ADAMTS13-mediated cleavage. In order to determine whether the loss of high molecular weight (HMW) multimers associated with 2B VWD occurs due to the increased ADAMTS13 susceptibility or due to the increased binding to platelet GPIbα, we created a modified type 2B mouse model, in which the mice expressed ADAMTS13-insensitive 2B VWF. Methods: Three common type 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf (mVWF) cDNA sequence containing the ADAMTS13 cleavage site knockout, Y1605A/M1606A (CSKO). Recombinant mutant mVWF was produced using transient transfection of HEK293T cells. ADAMTS13 digestion of the recombinant mVWF was performed using a Tris-Urea system. Platelet binding affinity was assessed using a botrocetin-induced platelet-binding ELISA assay. 8–9 week old C57Bl6 VWF KO mice were hydrodynamically injected with 100 μ g of naked plasmid DNA containing the liver specific ET promoter and wildtype (WT) or mutant mouse Vwf DNA. The mice were sampled weekly; complete blood counts, VWF:Ag levels, VWFpp levels, and VWF multimer structure were examined. Results: Following transient transfections, mutant and wildtype mVWF were secreted at similar levels, with a full range of VWF multimer sizes. Recombinant 2B/CSKO mVWF is markedly ADAMTS13 cleavage insensitive, with >80 U mADAMTS13 required for 50% cleavage (WT mVWF requires 1 U mADAMTS13). Similar to 2B VWF, 2B/CSKO mVWF showed enhanced platelet binding affinity in the presence of botrocetin. Hydrodynamic injections of the naked expression plasmids did not result in adverse events in the mice. Mean 2B/CSKO platelet counts at day 7 were significantly reduced compared to WT platelet counts (149 and 370, respectively). At day 14, only the mice expressing V1316M/CSKO remained thrombocytopenic, with platelet counts similar to those seen with the V1316M mutation alone (170 and 171, respectively). By day 14, the mice expressing 2B/CSKO VWF had significantly lower mean VWF:Ag levels than WT mice or mice expressing the CSKO alone (see table below). The mean VWFpp/VWF:Ag ratios were significantly increased in the 2B/CSKO mice when compared to the WT mice (1.53 and 1.00, respectively). There was no loss of HMW multimers in the mice expressing R1306W/CSKO and R1341Q/CSKO and, indeed, these mice showed significantly more multimer bands than WT mice (days 7 to 21; 14.5±0.6 and 13.9±1.2 multimer bands versus 10.2±0.5 bands for WT). In marked contrast, the mice expressing V1316M/CSKO VWF showed complete loss of high molecular weight multimers, similar to mice expressing the same 2B VWF mutation alone (8.3±1.8 and 7.7±0.9 multimer bands, respectively). Conclusions: Mice expressing R1306W/CSKO and R1341Q/CSKO VWF have an absence of thrombocytopenia and show the presence of supranormal high molecular weight VWF multimer bands. These results suggest that enhanced ADAMTS13-mediated cleavage plays an important role in the type 2B phenotype associated with these mutations. In contrast, low V1316M/CSKO VWF:Ag was associated with both thrombocytopenia and loss of HMW multimers, demonstrating that enhanced GPIbα binding affinity is the predominant mechanism associated with this mutation. Thus, the importance of ADAMTS13 susceptibility in 2B VWD is mutation-dependent, and ADAMTS13-mediated cleavage plays an important role in enhancing the severity of the type 2B VWD phenotype associated with the R1306W and R1341Q mutations. Disclosures: No relevant conflicts of interest to declare.

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Classification and Characterization of Hereditary Types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (Unclassifiable) von Willebrand Disease

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029606293422 ◽

2006 ◽

Vol 12 (4) ◽

pp. 397-420 ◽

Cited By ~ 26

Author(s):

Jan Jacques Michiels ◽

Zwi Berneman ◽

Alain Gadisseur ◽

Marc van der Planken ◽

Wilfried Schroyens ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Bleeding Time ◽

Autosomal Dominant ◽

Von Willebrand Disease ◽

Dominant Type ◽

Prolonged Bleeding Time ◽

The Individual ◽

Von Willebrand ◽

High Molecular Weight Multimers

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

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Two novel type 2N von Willebrand disease–causing mutations that result in defective factor VIII binding, multimerization, and secretion of von Willebrand factor

Blood ◽

10.1182/blood.v95.6.2000 ◽

2000 ◽

Vol 95 (6) ◽

pp. 2000-2007 ◽

Cited By ~ 45

Author(s):

Simon Allen ◽

Adel M. Abuzenadah ◽

Joanna L. Blagg ◽

Joanna Hinks ◽

I. Mandy Nesbitt ◽

...

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

High Molecular Weight ◽

Von Willebrand Factor ◽

Amino Acid Position ◽

Von Willebrand Disease ◽

Mild Reduction ◽

Von Willebrand ◽

Willebrand Factor ◽

High Molecular Weight Multimers

Abstract Two novel mutations, a T-to-C transition at nucleotide 2612 and a T-to-G transversion at nucleotide 3923 of the von Willebrand factor (vWF) complementary DNA, were detected by analysis of the vWF gene in DNA from members of 2 families with atypical von Willebrand disease. The T2612C transition predicts substitution of cysteine by arginine at amino acid position 788 (C788R), and the T3923G transversion predicts substitution of cysteine by glycine at position 1225 (C1225G) of pre-pro-vWF. The patients homozygous for the C788R and C1225G mutations both had a partial vWF deficiency (0.18 IU/mL and 0.07 IU/mL vWF antigen, respectively); vWF in plasma from patients homozygous for either the C788R or the C1225G mutation failed to bind factor VIII and lacked high molecular weight multimers. Recombinant (r) vWF molecules having the C788R or C1225G mutation were expressed in COS-7 cells. Both rvWF C788R and rvWF C1225G exhibited significantly impaired secretion and failed to bind factor VIII. Recombinant vWF C788R in COS-7 culture medium showed a severe reduction in high molecular weight multimers, whereas rvWF C1225G showed a very mild reduction in high molecular weight multimers when compared with wild-type rvWF.

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Circulating Von Willebrand factor and high molecular weight multimers as markers of endothelial injury predict COVID-19 in-hospital mortality

Angiogenesis ◽

10.1007/s10456-020-09762-6 ◽

2021 ◽

Author(s):

Aurélien Philippe ◽

Richard Chocron ◽

Nicolas Gendron ◽

Olivier Bory ◽

Agathe Beauvais ◽

...

Keyword(s):

Molecular Weight ◽

Hospital Mortality ◽

High Molecular Weight ◽

Von Willebrand Factor ◽

Endothelial Injury ◽

Von Willebrand ◽

Willebrand Factor ◽

High Molecular Weight Multimers

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A novel von Willebrand disease–causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion

Blood ◽

10.1182/blood.v96.2.560 ◽

2000 ◽

Vol 96 (2) ◽

pp. 560-568 ◽

Cited By ~ 41

Author(s):

Simon Allen ◽

Adel M. Abuzenadah ◽

Joanna Hinks ◽

Joanna L. Blagg ◽

Turkiz Gursel ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Von Willebrand Factor ◽

Family Members ◽

Amino Acid Position ◽

Von Willebrand Disease ◽

Molecular Defect ◽

Wild Type ◽

Von Willebrand ◽

Willebrand Factor

Abstract In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

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A Revised Classification of von Willebrand Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642471 ◽

1994 ◽

Vol 71 (04) ◽

pp. 520-525 ◽

Cited By ~ 378

Author(s):

J Evan Sadler

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Dependent Function ◽

Severe Deficiency ◽

Partial Deficiency ◽

Deficiency Type ◽

Von Willebrand

SummaryA simplified phenotypic classification of von Willebrand disease is proposed that is based on differences in pathophysiology. Quantitative defects arc divided into partial deficiency (type 1) and severe deficiency (type 3). Qualitative defects (type 2) are divided into four subcategories. Type 2A refers to variants with decreased platelet-dependent function associated with the loss of high-molecular weight VWF multimers. Type 2B refers to variants with increased affinity for platelet glycoprotein lb. Type 2M refers to qualitatively abnormal variants with decreased platelet-dependent function not associated with the loss of high-molecular weight multimers. Type 2N refers to variants with decreased affinity for factor VIII. When recognized, mixed phenotypes caused by compound heterozygosity are indicated by separate classification of each allele. Standard amino acid and nucleotide numbering schemes are recommended for the description of mutations.

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