Impaired dimerization of von Willebrand factor subunit due to mutation A2801D in the CK domain results in a recessive type 2A subtype IID von Willebrand disease

2006 ◽  
Vol 95 (05) ◽  
pp. 776-781 ◽  
Author(s):  
Antoine Hommais ◽  
Alain Stépanian ◽  
Edith Fressinaud ◽  
Claudine Mazurier ◽  
Katia Pouymayou ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Resolution ◽  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Full Range ◽  
Von Willebrand Disease ◽  
Recessive Type ◽  
Low Levels ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe CK domain of von Willebrand factor (VWF) is involved in the dimerization of the protein.We identified the homozygous substitution A2801D of the CK domain in two siblings. Patients had low levels of VWF in plasma, abnormal ristocetin-induced binding to platelets and abnormal multimeric pattern witha lack of high molecular weight (HMW) forms and the presence of intervening bands between normal multimers. Accordingly, they were classified in type 2A, subtype IID, von Willebrand disease (VWD). Both asymptomatic parents carried the mutation at the heterozygous state.Their plasmaVWF exhibited the full range of multimers found in normal plasma.When analyzed by high resolution gel electrophoresis, very faint bands corresponding to the position of intervening bands of the propositus can be observed. The mutated recombinant (r)VWF-D2801, the hybrid rVWF-A/D2801 and the mutated C-terminal VWF fragment rSPII-D2801 were expressed in COS-7 cells. rVWF-D2801 showed an abnormal multimeric distribution similar to that of the propositus’VWF with intervening bands and a lack of HMW species. rVWF-A/D2801 exhibited the full range of multimers and the aberrant sized forms observed both in propositus’VWF and in rVWF-D2801. rSPII-WT assembled correctly into a dimer of 220 kDa. rSPII-D2801 appeared as a mixture of monomeric and dimeric forms which may be related to the abnormal multimeric pattern of the propositus and both mutated rVWF. We concluded that mutation A2801D disturbs the folding of the CK domain, which may result in a mixture of monomers and dimers of VWF. Multimers containing either an odd or even number of mature subunits are produced, and the presence of monomers appears to limit the degree of multimerization. In the heterozygousVWF,the presence of normal dimers improves the multimerization process. In conclusion, the mutation A2801D appears to be responsible fora recessive type 2A, subtype IID, VWD.

The Genetic Defect of Type I von Willebrand Disease “Vicenza” Is Linked to the von Willebrand Factor Gene

1993 ◽  
Vol 69 (02) ◽  
pp. 173-176 ◽  
Author(s):  
Anna M Randi ◽  
Elisabetta Sacchi ◽  
Gian Carlo Castaman ◽  
Francesco Rodeghiero ◽  
Pier Mannuccio Mannucci
Keyword(s):  
Von Willebrand Factor ◽  
Autosomal Dominant ◽  
Genetic Defect ◽  
Von Willebrand Disease ◽  
Type I ◽  
Bleeding Tendency ◽  
Factor Gene ◽  
Low Levels ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType I von Willebrand disease (vWD) Vicenza is a rare variant with autosomal dominant transmission, characterized by the presence of supranormal von Willebrand factor (vWF) multimers in plasma, similar to those normally found in endothelial cells and megakaryocytes. The patients have very low levels of plasma vWF contrasting with a mild bleeding tendency. The pathophysiology of this subtype is still unknown. The presence of supranormal multimers in the patients’ plasma could be due to a mutation in the vWF molecule which affects post-translational processing, or to a defect in the cells’ processing machinery, independent of the vWF molecule. In order to determne if type I vWD Vicenza is linked to the vWF gene, we studied six polymorphic systems identified within the vWF gene in two apparently unrelated families with type I vWD Vicenza. The results of this study indicate a linkage between vWF gene and the type I vWD Vicenza trait. This strongly suggests that type I vWD Vicenza is due to a mutation in one of the vWF alleles, which results in an abnormal vWF molecule that is processed to a lesser extent than normal vWF.

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

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 560-568 ◽  
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.

scholarly journals New variant of von Willebrand disease type II with markedly increased levels of von Willebrand factor antigen and dominant mode of inheritance: von Willebrand disease type IIC Miami

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 169-175 ◽  
Author(s):  
MR Ledford ◽  
I Rabinowitz ◽  
JE Sadler ◽  
JW Kent ◽  
F Civantos
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Bleeding Time ◽  
Autosomal Dominant ◽  
Disease Type ◽  
Von Willebrand Disease ◽  
Dominant Inheritance ◽  
New Variant ◽  
Von Willebrand ◽  
Willebrand Factor

A variant of von Willebrand disease (vWD) was identified in six members of a kindred spanning four generations. The proband was a 46-year-old woman with a lifelong history of bleeding, a prolonged bleeding time (> 15 minutes), markedly elevated von Willebrand factor (vWF) antigen (vWF:Ag = 2.09 U/mL), slightly reduced ristocetin cofactor activity, and a plasma vWF multimer pattern similar to that of vWD type IIC. Similar findings were observed in her three children, mother, and brother. In affected family members, platelet and plasma vWF multimer patterns were discrepant with higher molecular weight multimers observed in platelet vWF. Following a 1-Des-amino-8-D-arginine vasopressin (DDAVP) challenge, the proband failed to normalize her bleeding time even though vWF: Ag rose by 70% and higher molecular weight multimers were increased slightly. Genetic studies were consistent with autosomal dominant inheritance of a mutation within the vWF gene. By sequencing of cloned genomic DNA, mutations were excluded in exons 4, 5, 14, and 15, which encode regions of the vWF propeptide proposed to be important in multimer biosynthesis. Mutations also were excluded in exons 28 to 31, which encompass the known mutations that cause vWD types IIA, IIB, and B. This new variant of vWD, characterized by autosomal dominant inheritance, a qualitative defect that resembles vWD type IIC, and increased plasma vWF:Ag, was tentatively designated vWD type IIC Miami.

ADAMTS13 In 4 Different VWF/VIII Concentrates and Its Impact on Therapy.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3677-3677
Author(s):  
Mirjeta Qorraj ◽  
Tanja Falter ◽  
Sarah Steinemann ◽  
Thomas Vigh ◽  
Inge Scharrer
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Relative Reduction ◽  
Hemorrhagic Diathesis ◽  
Adamts13 Activity ◽  
Kinetic Measurements ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3677 Introduction: The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates. Methods: We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers. Results: The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level. Conclusion: All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The roles of von Willebrand factor and factor VIII in arterial thrombosis: studies in canine von Willebrand disease and hemophilia A

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2644-2651 ◽  
Author(s):  
TC Nichols ◽  
DA Bellinger ◽  
RL Reddick ◽  
SV Smith ◽  
GG Koch ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Arterial Thrombosis ◽  
Hemophilia A ◽  
Full Range ◽  
Von Willebrand Disease ◽  
Arterial Stenosis ◽  
Canine Plasma ◽  
Von Willebrand ◽  
Willebrand Factor

We have studied the roles of von Willebrand factor (vWF) and factor VIII in arterial thrombosis in four canine phenotypes: normal (n = 6), hemophilia A (n = 11), von Willebrand disease (vWD) (n = 9), and hemophilia A/vWD (n = 1). vWF activity was determined by botrocetin- induced agglutination of fixed human platelets and vWF antigen (vWF:Ag) by Laurell electroimmunoassay and crossed immunoelectrophoresis. Plasma from normal dogs and those with hemophilia A had vWF activity, vWF:Ag, and a full range of vWF:Ag multimers on gel electrophoresis equivalent to normal canine plasma pool. Platelet cytosol contents were isolated by freezing and thawing, triton X-100 solubilization, or sonication of washed platelets with and without protease inhibitors and inhibitors of platelet activation. Washed platelets were also stimulated with calcium ionophore and MgCl2. There was no measurable vWF activity or vWF:Ag in platelet lysates or releasates in any dog regardless of phenotype. All dogs were studied using a standard arterial stenosis and injury procedure to induce arterial thrombosis. Thromboses were detected by cyclic reductions in Doppler blood flow velocity. Vessels were examined by light and scanning electron microscopy. Thrombosis developed in the arteries of normal (9 of 10) and hemophilia A dogs (16 of 16) but in none of the vWD dogs (0 of 10). Infusion of canine vWF cryoprecipitate into vWD dogs markedly shortened bleeding time but did not support thrombosis as seen in dogs with vWF in the plasma and subendothelium. Thrombosis, then, fails to occur when vWF is absent from the plasma and subendothelial compartments or present only in the plasma compartment. These data are consistent with the hypothesis that vWF in the plasma and subendothelium supports thrombosis. Neither plasma FVIII nor platelet vWF is essential for thrombosis in this model.

Effect of Type IIB von Willebrand Disease Mutation Arg(545)Cys on Platelet Glycoprotein Ib Binding – Studies with Recombinant von Willebrand Factor

1993 ◽  
Vol 70 (06) ◽  
pp. 1058-1062 ◽  
Author(s):  
Aida Inbal ◽  
Nurit Kornbrot ◽  
Paul Harrison ◽  
Anna M Randi ◽  
J Evan Sadler
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Wild Type ◽  
Binding Studies ◽  
Function Type ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein lb (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristic gain-of-function type IIB vWD phenotype.

ADAMTS-13 Binds Platelets in a Specific, Divalent Cation and Activation Dependent Manner.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2657-2657
Author(s):  
Andrea Artoni ◽  
Isabella Garagiola ◽  
Rossana Lombardi ◽  
Flora Peyvandi ◽  
Pier Mannuccio Mannucci
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Divalent Cations ◽  
Von Willebrand Disease ◽  
Normal Donor ◽  
Dependent Manner ◽  
Platelet Surface ◽  
Low Levels ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS-13 cleaves high molecular weight von Willebrand factor (VWF) realeased by the endothelium in order to prevent massive intravascular platelets adhesion and aggregation as pathologically observed in thrombotic thrombocytopenic purpura. ADAMTS-13 is present at low levels in plasma. We therefore surmised that platelets are able to specifically bind the metalloprotease on their surface, hereby concentrating the enzyme where it is most required. With this as background, 96 wells polystyrene NUNC plates were coated either with albumin or fibrinogen or VWF or recombinant ADAMTS-13 (each at 10 μg/ml) and then blocked with 5% albumin overnight. Washed platelets (100,000/μl), incubated with divalent cations, were then let adhere to the wells for 1 hr at 37°C. After extensive washing adherent platelets were lysed, p-nitrophenilphosphate was added and the reaction was stopped with NaOH 2M. Detection was done by assessing optical density at 405 nm. Binding of washed platelets preincubated with 2mM CaCl2 and/or 2mM MgCl2 to wells covered with immobilized recombinant ADAMTS-13 was significantly higher than binding to wells coated with albumin (p<0.001), was at the same levels of the binding to wells covered with recombinant VWF and approximately half of binding to the wells coated with fibrinogen. When washed platelets were preincubated with EDTA 2mM binding to the wells coated with fibrinogen or recombinant ADAMTS-13 decreased at the same degree of the wells covered with albumin. Preincubation of platelets with antibodies against αIIbβ3 (7E3, 10 μg/ml) or GpIb (10 μg/ml) did not effect the levels of binding to recombinant ADAMTS-13 while the binding to fibrinogen and VWF was totally abolished. Activation of platelets, obtained by preincubating platelets with ADP (5 μM) or collagen (10 μg/ml), significantly increased their binding to recombinant ADAMTS-13 (p<0.001) as compared to the binding of non-activated platelets. Immunofluorescent studies were then performed to see whether or not ADAMTS-13 bound to the platelet plasma membrane using as primary antibody a murine anti-human ADAMTS-13 monoclonal antibody (13E2). A positive membrane fluorescent signal was detected using as a source of platelets either a normal donor or a patient with type III Von Willebrand disease, demonstrating that ADAMTS-13 is located on the platelet surface independently from VWF. In conclusion ADAMTS-13 binds to the surface of platelets, the binding is specific, activation and divalent cations dependent, inhibited by EDTA, and not mediated by VWF on the membrane of platelets; the binding site does not appear to be αIIbβ3 or GpIb. Hence it can be hypothesized that ADAMTS-13 on platelet membrane might cleave high molecular weight VWF.

Recombinant Human Von Willebrand Factor (rhVWF): First-In-Human Study Evaluating Pharmacokinetics, Demonstrating Safety and Tolerability In Type 3 Von Willebrand Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 237-237 ◽  
Author(s):  
Tobias Suiter ◽  
Michael Laffan ◽  
Pier M. Mannucci ◽  
Christine L. Kempton ◽  
Edward H Romond ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Phase 1 ◽  
Von Willebrand Disease ◽  
Fviii Activity ◽  
Post Infusion ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 237 Von Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF is the largest soluble multimeric plasma glycoprotein, which facilitates platelet aggregation and stabilizes FVIII in the circulation. Patients with type 3 disease display severe hemorrhagic symptoms, mainly in mucosal tissues, muscle and joints. Replacement of VWF stabilizes endogenous FVIII to hemostatic levels within hours. Commercially available VWF/FVIII concentrates are plasma-derived (pd) and subject to limitations such as donor dependency, risk of blood-borne pathogen transmission, lack of high molecular weight VWF multimers, and variation in multimer composition. A novel recombinant human VWF (rhVWF) has been developed using a plasma-free method, which represents the largest protein ever produced using recombinant technology. Safety, tolerability and pharmacokinetics of the rhVWF combined at a fixed ratio with rFVIII were investigated in a Phase 1 multicenter, international clinical study in 31 patients with type 3 VWD and severe type 1 VWD. Four concentrations of rhVWF (2, 7.5, 20 and 50 IU VWF:RCo/kg) were administered in a dose-escalating manner in separate cohorts. rhVWF was well tolerated, and no thrombotic events, VWF inhibitors or other serious adverse reactions were observed. Pharmacokinetics of rhVWF/rhFVIII (50 IU VWF:RCo/kg and 38.5 IU FVIII/kg) compared with pdVWF/pdFVIII (50 IU VWF:RCo/kg and 21 IU/kg FVIII/kg) were evaluated in a sub-group of 8/31 patients using a randomized, crossover design (8-day minimum washout period). Interim data in 8 subjects show a higher degree of secondary FVIII activity with rhVWF/rhFVIII compared to pdVWF/pdFVIII (see Figure 1) that is not solely due the difference in the rhVVF:FVIII infusion ratio (1.3:1 rhVWF/rhFVIII vs. approximately 2:1 pdVWF/pdFVIII). The pharmacokinetics of the rhVWF:RCo and pdVWF:RCo were comparable and were also reflected in the VWF:Ag and collagen binding activity. Evidence is also provided for the in vivo cleavage of the ultra-high molecular weight multimers of rhVWF by endogenous ADAMTS13. In summary, interim data from the ongoing Phase 1 study, demonstrate that rhVWF is safe and well tolerated, has VWF:RCo pharmacokinetics that are comparable to pdVWF and enhances stabilization of endogenous FVIII. Multiple doses of rhVWF/rhFVIII would be expected to have beneficial effects in major surgery and severe mucosal bleeding events. These data would also support the treatment concept to administer rhVWF alone once a therapeutic baseline level of endogenous FVIII is achieved (after 1–2 doses).Figure 1:Preliminary PK data from 8 subjects post-infusion of either rhVWF/rhFVIII or pdVWF/pdFVIII. Endogenous FVIII activity reached a plateau after 6 hours and remained stable for at least 30 hours. FVIII was still elevated well above baseline at 96 hoursFigure 1:. Preliminary PK data from 8 subjects post-infusion of either rhVWF/rhFVIII or pdVWF/pdFVIII. Endogenous FVIII activity reached a plateau after 6 hours and remained stable for at least 30 hours. FVIII was still elevated well above baseline at 96 hours Disclosures: Suiter: Baxter BioScience: Employment. Laffan:Baxter BioScience: Consultancy. Mannucci:Baxter BioScience: Consultancy. Kempton:Baxter BioScience: Consultancy. Romond:Baxter BioScience: Consultancy. Shapiro: Baxter BioSci- ence: Consultancy. Birschmann:Baxter BioScience: Consultancy. Gill:Baxter BioScience: Consultancy. Ragni:Baxter BioScience: Consultancy. Turecek:Baxter BioScience: Employment. Ewenstein:Baxter Bioscience: Employment. Baxter BioScience:Baxter BioScience: Employment.

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

Blood ◽  
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.

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

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 560-568 ◽  
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

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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