von Willebrand Factor Activity Detected in a Monoclonal Antibody-based ELISA: an Alternative to the Ristocetin Cofactor Platelet Agglutination Assay for Diagnostic Use

1997 ◽  
Vol 78 (04) ◽  
pp. 1272-1277 ◽  
Author(s):  
Paul J Murdock ◽  
Barry J Woodhams ◽  
Kathy B Matthews ◽  
K John Pasi ◽  
Alison H Goodall
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Polyclonal Antibody ◽  
Solid Phase ◽  
Laboratory Diagnosis ◽  
Capture Antibody ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

SummaryThe monoclonal antibody RFF-VIII:R/1 recognises an epitope on von Willebrand factor involved in its interaction with GPIbα. A two-site, solid phase ELISA has been established using RFF-VIII:R/1 as the solid-phase, capture antibody and an enzyme-conjugated, polyclonal antibody to human VWF, which provides an assay for VWF functional activity with a detection limit of 0.5 U/dl VWF and an interassay %CV<10. Plasma from 192 VWD patients (48 studied retrospectively; 144 prospectively) showed VWF levels of <50 U/dl in type 1 patients (n = 156), <25 U/dl in type 2A (n = 26) and <35 U/dl in type 2B (n = 8) which, in type 1 and 2A patients, correlated with RiCoF activity (r >0.82). In plasma from patients with type 1 VWD values of VWF in the Mab-based ELISA were similar to levels of VWF:Ag measured in a polyclonal antibody-based ELISA (r >0.87) but were significantly lower than VWF:Ag in type 2A and 2B plasmas (p <0.0005), allowing discrimination of variant VWD. The Mab-based ELISA has advantages of sensitivity and reproducibility over the RiCoF assay to measure VWF activity and can be used to analyse stored samples. In conjunction with an ELISA for VWF:Ag and VWF multimer analysis, it provides a reliable method, for the laboratory diagnosis of VWD.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

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.

A Two-Site, Monoclonal Antibody-Based Immunoassay for von Willebrand Factor -Demonstration that vWF Function Resides in a Conformational Epitope

1986 ◽  
Vol 55 (03) ◽  
pp. 318-324 ◽  
Author(s):  
S Chand ◽  
A McCraw ◽  
R Hutton ◽  
E G D Tuddenham ◽  
A H Goodall
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Solid Phase ◽  
Fluid Phase ◽  
Conformational Epitope ◽  
Type Ii ◽  
Normal Individuals ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Umbilical Cord Vein

SummaryTwo monoclonal antibodies (RFF-VIII: R/l and RFF-VIII:R/ 2) which recognise the same epitope on von Willebrand factor (vWF) have been used in a simple, two-site, solid-phase immunoradiometric (IRMA) or enzyme-linked assay (ELISA) to analyse vWF in plasma from normal individuals and from patients with von Willebrand’s disease (vWD). Results obtained confirm our previous findings (using RFF-VIII :R/2 in a one-site, fluid-phase IRMA) that the MAbs detect the presence of an epitope on the vWF molecule that reflects its function. This epitope is involved in vWF binding to the GPIb protein on platelets. It is reduced in all types of vWD, including type II (or variant) vWD. It is present in normal plasma, in vWF released from normal platelets and from cultured umbilical cord vein endothelial cells. The epitope is, however, found to be reduced in serum. Studies on SDS-treated vWF prove that this GPIb-binding site is dependent on the conformation of the vWF multimers.

Type 2M vWD Resulting from a Lysine Deletion within a Four Lysine Residue Repeat in the A1 Loop of von Willebrand Factor

2000 ◽  
Vol 84 (08) ◽  
pp. 188-194 ◽  
Author(s):  
P. V. Jenkins ◽  
C. Gaucher ◽  
E. Meriane ◽  
P. W. Collins ◽  
K. J. Pasi ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Lysine Residue ◽  
Antigen Level ◽  
The Uk ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

SummaryType 1 von Willebrand disease is characterized by a decreased plasma concentration of functionally normal von Willebrand factor (vWF) whereas type 2M is characterised by an abnormal vWF displaying decreased affinity for platelets. In these two types of patients, the multimeric structure of vWF is normal.We report here the identification, in two unrelated families from the UK and Algeria, of an in-frame 3 bp deletion, at the heterozygous state, resulting in the deletion of a lysine residue within a four lysine repeat at position 642-645 of the mature vWF subunit (del K1405-1408 in prepro vWF). The patients who have a discrepancy between vWF antigen level and vWF ristocetin cofactor activity exhibited decreased ristocetin-induced binding but only a slight decrease in the percentage of high molecular weight (HMW) multimers in plasma.Recombinant vWF harbouring this deletion did not bind to platelet GPIb in the presence of ristocetin or botrocetin although the protein is multimerized. Consequently, this lysine deletion was considered as a type 2M vWD mutation.

scholarly journals Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3622-3630 ◽  
Author(s):  
C Denis ◽  
JA Williams ◽  
X Lu ◽  
D Meyer ◽  
D Baruch
Keyword(s):  
Monoclonal Antibody ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Solid Phase ◽  
Rgd Sequence ◽  
Alpha V Beta 3 ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Endothelial Cell Adhesion

Abstract The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744–1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1–1365) and SpII (residues 1366–2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD- containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.

Evaluation of commercial von Willebrand factor collagen binding assays to assist the discrimination of types 1 and 2 von Willebrand disease

2010 ◽  
Vol 104 (11) ◽  
pp. 1009-1021 ◽  
Author(s):  
Emmanuel Favaloro
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Binding Assays ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Control Samples

SummaryThis study reports on the evaluation of seven commercial von Wille-brand factor (VWF) collagen binding (VWF:CB) assays to potentially assist the discrimination of types 1 and 2 von Willebrand disease (VWD). Samples from 25 patients with type 1 VWD, of varying severity, were co-tested with 16 samples from patients with types 2A or 2B VWD, plus various control samples, using each commercial VWF:CB assay assessed against our standard (reference) in-house VWF:CB assay, as well as our in-house VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) assays. Commercial VWF:CB assays varied in their ability to discriminate types 1 and 2A/2B VWD. The optimal VWF:CB/VWF:Ag ratio at which optimal discrimination occurred also differed between assays, with some improvements observed with some (but not all) as-says following a harmonisation process that aimed to correct for different calibrator effects. Assay variability also compromised assay utility in some test occasions. Future standardisation and improvements in some commercial VWF:CB assays are needed before the VWF:CB assay can be more fully and globally utilised for discrimination of VWD types in diagnostic laboratories.

scholarly journals Adhesive protein expression on thrombin-stimulated platelets: time- dependent modulation of anti-fibrinogen, -fibronectin, and -von Willebrand factor antibody binding

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 948-953 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Polyclonal Antibody ◽  
Time Course ◽  
Surface Membrane ◽  
Membrane Receptors ◽  
Time Dependent ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Platelets contain a pool of endogenous adhesive proteins that can be released and may bind to surface membrane receptors under appropriate conditions. Because the binding of exogenous fibrinogen to platelets was shown previously to be accompanied by a time-dependent decrease in fibrinogen accessibility to antibody and enzymes, studies were performed to evaluate changes in the expression of endogenous fibrinogen released from thrombin-stimulated platelets using monospecific polyclonal and monoclonal antibody F(ab')2 fragments. Parallel studies were performed to compare the expression of released fibronectin and von Willebrand factor (vWF). Binding of polyclonal antibody F(ab')2 fragments directed against individual adhesive proteins was inhibited by EDTA or the 10E5 monoclonal antibody, suggesting that fibrinogen, fibronectin, and vWF expression was mediated, in large part, by divalent cation-dependent interactions with the glycoprotein IIb-IIIa complex. Interestingly, when polyclonal antibody F(ab')2 fragments were added to platelet suspensions at discrete times after thrombin stimulation, antifibrinogen F(ab')2 binding decreased by 72% +/- 15% (mean +/- SD, n = 22) over a 60-minute time course, whereas antifibronectin and anti-vWF antibody F(ab')2 fragment binding changed minimally (6% +/- 23%, n = 22 and 3% +/- 26%, n = 14, respectively). Similar observations were made with monoclonal antibodies. Parallel experiments using 125I-labeled fibrinogen as a marker indicated that the observed decrease in antifibrinogen F(ab')2 binding was not accompanied by fibrinogen dissociation. Moreover, antibody accessibility to platelet-bound fibrinogen could be restored after Triton X-100 platelet lysis. The data suggest that fibrinogen, fibronectin, and vWF are not coordinately expressed on thrombin- stimulated platelets. Rather, fibrinogen expression appears transient compared with the expression of fibronectin and vWF. The ability of platelets to secrete and organize adhesive proteins on their surface is likely to have important implications for hemostasis and thrombosis.

Resemblance to vWD Types and Laboratory Diagnosis of Obligatory Carriers of Type 3 von Willebrand Disease

2010 ◽  
Vol 17 (6) ◽  
pp. E21-E24
Author(s):  
Mehmet Akin ◽  
Deniz Yilmaz Karapinar ◽  
Can Balkan ◽  
Yilmaz Ay ◽  
Kaan Kavakli
Keyword(s):  
Von Willebrand Factor ◽  
Laboratory Diagnosis ◽  
Von Willebrand Disease ◽  
Fast Method ◽  
Close Relatives ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Severe Type

Objectives: It is important to diagnose obligatory carrier (OC) type 3 von Willebrand Disease (vWD) in countries, such as Turkey, where marriages between relatives is common. However, mild bleeding or no bleeding in such patients complicates the diagnosis of the disease. It is not clear how the diagnosis of OC type 3 vWD will be made based on FVIII:C (Factor VIII activity), vWF:Ag (von Willebrand factor antigen), vWF:RCo (von Willebrand factor ristocetin cofactor activity), and PFA (platelet function analyzer )-100 parameters. Therefore, the purpose of the study is to investigate how OC type 3 vWD diagnoses may be established by studying laboratory phenotypes of close relatives of patients with diagnosed 3 vWD. Patients and Methods: 8 patients with type 3 vWD (index cases) and 20 patients who were defined as OCs type 3 vWD were enrolled into the study. Result: 10 cases had similarity with mild type VWD, 4 cases had similarity with moderate type 1 vWD, 4 other cases had type 1 or 2 vWD similarities, 1 case had similarity with severe type 1 vWD, and 1 case also had similarity with severe type 1 or type 2 vWD; regarding their laboratory phenotypic characteristics. Conclusion: we identified that OC type 3 vWD is similar specifically to type 1 vWD in terms of laboratory phenotypic character, and we suggest that it may be used with PFA-100 as an easy and fast method in screening relatives.

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