scholarly journals Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1738-1743 ◽  
Author(s):  
AS Ribba ◽  
JM Lavergne ◽  
BR Bahnak ◽  
A Derlon ◽  
G Pietu ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Functional Domains ◽  
Gene Coding ◽  
Gradient Gel Electrophoresis ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5′ end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.

scholarly journals Duplication of a methionine within the glycoprotein Ib binding domain of von Willebrand factor detected by denaturing gradient gel electrophoresis in a patient with type IIB von Willebrand disease

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1738-1743 ◽  
Author(s):  
AS Ribba ◽  
JM Lavergne ◽  
BR Bahnak ◽  
A Derlon ◽  
G Pietu ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Functional Domains ◽  
Gene Coding ◽  
Gradient Gel Electrophoresis ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand disease (vWD) type IIB is characterized by an increased reactivity of von Willebrand factor (vWF) with platelets and a lack of large multimers. Exon 28 of the vWF gene encodes for functional domains involved in the binding of vWF to GPIb, and it is presumed that the defects in type IIB vWD lie within or adjacent to these functional domains. We screened overlapping DNA fragments generated by the polymerase chain reaction (PCR) that spanned the 1,379 bp of exon 28 of a type IIB vWD patient using denaturing gradient gel electrophoresis (DGGE). To increase the power of DGGE to detect base changes, we used the PCR to attach a G + C-rich sequence. In the type IIB patient, a DNA fragment at the 5′ end of exon 28 demonstrated homoduplex and heteroduplex complexes after DGGE, a pattern characteristic of heterozygous genes after melting and reannealing during the PCR. Sequencing of the cloned insert from the patient showed a duplication of an ATG in one gene coding for a Met at amino acids 540 to 541 in the mature vWF subunit. This duplication leads to three consecutive methionines in the patient's sequence. The duplicated Met resides within a disulfide bond loop proposed to be important in the function of the GPIb binding domain of vWF. The patient's nephew, who also has type IIB vWD, showed the same duplicated codon, linking the defect to the abnormal phenotype in this family. These nucleotide changes were not found in 100 chromosomes analyzed either by DGGE or hybridization with an allele specific oligonucleotide containing the duplicated ATG codon. In addition, the same oligonucleotide hybridized only to DNA from type IIB vWD individuals and not to DNA from normal members of the family. Therefore, we conclude that this duplicated Met modifies the GPIb binding domain of vWF and causes type IIB vWD in this family.

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 defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor

Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 1013-1021 ◽  
Author(s):  
PA Kroner ◽  
PA Foster ◽  
SA Fahs ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Solid Phase ◽  
Disease Patient ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Mutant Proteins ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
Willebrand Factor

In this report we describe the further investigation of the von Willebrand factor (vWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by one-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing recombinant FVIII. Sequence analysis of the FVIII-binding domain encoded by the vWF mRNA of the affected individual identified mutations in both vWF alleles. In allele A, the mutations C2344T and T2451A result in the substitution of Trp for Arg19 (R19W) and of G1n for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported polymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding domain (delta G2515) that results in the premature termination of translation. Analysis of the binding of FVIII by full- length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q substitutions are the cause of the defective vWF/FVIII interaction in this patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins are recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12, and 31H3, which block the binding of FVIII by vWF, indicating that amino acids Arg19, Thr26, and His54 are not critical residues in the epitopes of these antibodies.

scholarly journals Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2322-2328 ◽  
Author(s):  
KA Cooney ◽  
D Ginsburg
Keyword(s):  
Von Willebrand Factor ◽  
Binding Domain ◽  
Von Willebrand Disease ◽  
Surface Glycoprotein ◽  
Platelet Surface ◽  
Disease Mutations ◽  
A Domains ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete “on” or “off” conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains.

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.

HOMOZYGOUS AND HETEROZYGOUS COMPLETE DELETIONS OF THE VON WILLEBRAND FACTOR GENE CODING REGION IN SEVERE VON WILLEBRAND DISEASE AND CARRIERS

1987 ◽  
Author(s):  
K Y Ngo ◽  
D Lynch ◽  
J Gitscher ◽  
N Ciavarella ◽  
Z Ruggeri ◽  
...  
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Genomic Dna ◽  
Untranslated Region ◽  
Gene Dosage ◽  
Von Willebrand Disease ◽  
Coding Region ◽  
Gene Coding ◽  
Von Willebrand ◽  
Willebrand Factor

Severe von Willebrand disease (vWD) is characterized by undetectable levels of von Willebrand factor (vWF), or trace amounts, in plasma and tissue stores. We have studied the genomic DNA of ten affected individuals from five families with this disorder using two cDNA probes. One probe extended from 175 base pairs of the 5’ untranslated region to the nucleotides encoding amino acid 618 of pro-vWF; the second extended from the nucleotides encoding amino acid 2225 of pro-vWF to 100 bp into the 3’ untranslated region. Three variants of the disorder were identified. Southern blots of restriction endonuclease digests and slot blots of undigested genomic DNA showed complete homozygous deletion of the vWF gene coding region in four affected siblings, three of whom had developed allo-antibodies. Gene dosage analysis performed with slot blots and laser densitometry were consistent with complete heterozygous deletions in both parents. The second variant was characterized by a complete heterozygous deletion of the vWF gene coding region in the propositus and one asymptomatic parent, suggesting that a different type of genetic abnormality was inherited from the other parent and that the patient was doubly heterozygous for distinct genetic abnormalities affecting vWF. In a third variant, no abnormalities could be detected. These techniques should prove useful in identifying carriers of severe vWD and also defining patients at risk of developing allo-antibodies to vWF.

Luminography – an Alternative Assay for Detection of von Willebrand Factor Multimers

1988 ◽  
Vol 60 (02) ◽  
pp. 133-136 ◽  
Author(s):  
R Schneppenheim ◽  
H Plendl ◽  
U Budde
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Detection Methods ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Luminescence Assay ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA luminescence assay was adapted for detection of von Willebrand factor multimers subsequent to SDS-agarose gel electrophoresis and electroblotting onto nitrocellulose. The method is as fast as chromogenic detection methods and appears to be as sensitive as autoradiography without the disadvantages of the latter.

Sign in / Sign up

Export Citation Format

Share Document