scholarly journals A Patient With Type 2N von Willebrand Disease Is Heterozygous for a New Mutation: Gly22Glu. Demonstration of a Defective Expression of the Second Allele by the Use of Monoclonal Antibodies

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3263-3269 ◽  
Author(s):  
J. Gu ◽  
S. Jorieux ◽  
J.M. Lavergne ◽  
C. Ruan ◽  
C. Mazurier ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Chinese Patient ◽  
Von Willebrand Disease ◽  
Expression Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Ability ◽  
New Mutation ◽  
Candidate Mutation ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractWe report the case of a Chinese patient who has subnormal von Willebrand factor (vWF ) level and normal vWF multimeric pattern, but a lack of vWF capacity to bind factor VIII (FVIII). Exons 18 to 20 of the patient's vWF gene were analyzed by DGGE and a G2354 → A substitution which changes the encoded amino acid sequence from Gly22 to Glu was identified. The patient is heterozygous for this substitution, creating a unique Sac I restriction site. Recombinant vWF (rvWF ) containing the candidate mutation was transiently expressed in COS-7 cells. It was processed and secreted normally but failed to bind FVIII. FVIII binding ability of hybrid rvWF, obtained by cotransfection of normal and mutated expression vectors and corresponding to a heterozygous genotype, was moderately decreased. To explain this functional discrepancy between patient's plasma vWF and hybrid rvWF, we used anti-vWF monoclonal antibodies (MoAbs) as capture in an enzyme-linked immunosorbent assay test. MoAb 32B12 recognized both wild-type and mutated rvWFs whereas MoAb 418 did not recognize mutated rvWF. Because MoAb 418 also failed to capture the plasma vWF from propositus, it means that his second nonmutated allele is not expressed or expressed at a very low level.

VON WILLEBRAND FACTOR (VWF) ANTIGENS IN URINE

1987 ◽  
Author(s):  
A M V Silveira ◽  
B Hessel ◽  
B Blombäck
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
High Performance ◽  
Molecular Size ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Normal Urine ◽  
Von Willebrand ◽  
Willebrand Factor

Human urine was analyzed using a sensitive enzyme linked immunosorbent assay (ELISA) for von Willebrand factor (VWF) antigen. Urine of healthy persons contained VWF immunoreactivity. In the urine of a patient with severe von Willebrand disease, the VWF antigen was only detectable after intravenous infusion of VWF-Factor VIII concentrate. The VWF antigen in normal urine was analyzed by gel permeation high performance liquid chromatography (HPLC) and gel electrophoresis combined with immunoblotting. These analyses revealed three immunoreactive components of Mr 350 kDa, 60 kDa, and 20 kDa, respectively, the 60 kDa being the major component. Monoclonal antibodies of known specificity to VWF molecule were used in ELISA and immunoblotting to analyze urinary VWF. The three components reacted with an antibody to the central part of VWF, which is called fragment I, and contains the binding site for collagen. No significant immunoreac-tion was observed with monoclonal antibodies to the Nor C-terminal portions of VWF.VWF derivatives of molecular size similar to the largest urinary antigens were also observed in normal plasma. However, there is not an obvious relationship between these plasma forms and the products in urine since reduction of plasma and urine yields different products.These results indicate that VWF antigens excreted in normal urine are most likely fragments of VWF produced by limited degradation in vivo. This degradation preserves the central part of VWF molecule, the one which reacts with the antibody that blocks the binding of VWF to collagen.

The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

1992 ◽  
Vol 68 (04) ◽  
pp. 464-469 ◽  
Author(s):  
Y Fujimura ◽  
S Miyata ◽  
S Nishida ◽  
S Miura ◽  
M Kaneda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Normal Individuals ◽  
Rag 1 ◽  
The One ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4862-4869 ◽  
Author(s):  
Mia Golder ◽  
Cynthia M. Pruss ◽  
Carol Hegadorn ◽  
Jeffrey Mewburn ◽  
Kimberly Laverty ◽  
...  
Keyword(s):  
Ferric Chloride ◽  
Von Willebrand Disease ◽  
Expression Vectors ◽  
Severe Thrombocytopenia ◽  
Normal Platelet ◽  
Injury Model ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbα binding. These “gain of function” mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF−/− mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride–induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride–induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

scholarly journals The von willebrand factor domain-mediating botrocetin-induced binding to glycoprotein IB lies between Val449 and Lys728

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 985-988 ◽  
Author(s):  
Y Fujimura ◽  
LZ Holland ◽  
ZM Ruggeri ◽  
TS Zimmerman
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Residue ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Tryptic Fragment ◽  
Alternative Agent ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Botrocetin, a component of Bothrops jararaca venom, induces von Willebrand factor (vWF)-dependent platelet agglutination and has been proposed as an alternative agent to ristocetin for evaluating vWF function. However, important differences between the vWF-platelet interactions induced by these two agents have suggested that different regions of vWF and the platelet may be involved in the interactions induced by the two agonists. We have recently demonstrated that binding of vWF to the platelet glycoprotein (GP) Ib receptor, either induced by ristocetin or as occurs spontaneously with asialo-vWF or vWF from IIb von Willebrand disease, is mediated by a domain residing on a 52/48- kilodalton (kD) tryptic fragment of vWF. This fragment extends from amino acid residue Val (449) to Lys (728). We have now found that this 52/48-kD fragment blocks botrocetin-induced binding of vWF to platelets and completely inhibits botrocetin-induced platelet agglutination. These results provide evidence that the vWF domain-mediating, botrocetin-induced platelet agglutination lies within the region delimited by this fragment and is therefore close to or identical with that which mediates ristocetin-induced binding and spontaneous binding of vWF to platelet GPIb. Anti-GPIb monoclonal antibodies also blocked agglutination, which showed that botrocetin, like ristocetin, induces binding of vWF to the GPIb receptor.

A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4663-4670 ◽  
Author(s):  
S. Jorieux ◽  
C. Gaucher ◽  
J. Goudemand ◽  
C. Mazurier
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Stop Codon ◽  
Novel Mutation ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequencing Analysis ◽  
Normal Platelet ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract 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.

scholarly journals A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2028-2033
Author(s):  
A Casonato ◽  
L De Marco ◽  
M Mazzucato ◽  
V De Angelis ◽  
D De Roia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Von Willebrand Disease ◽  
Bleeding Tendency ◽  
Binding Studies ◽  
Spontaneous Platelet Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.

Evidence for Extracellular Processing of Pro-von Willebrand Factor After Infusion in Animals With and Without Severe von Willebrand Disease

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1637-1647 ◽  
Author(s):  
P.L. Turecek ◽  
L. Pichler ◽  
W. Auer ◽  
G. Eder ◽  
K. Varadi ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Proteolytic Processing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Targeted Disruption ◽  
Agarose Gels ◽  
Before And After ◽  
Normal Human ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

A Novel Mutation in the D3 Domain of von Willebrand Factor Markedly Decreases Its Ability to Bind Factor VIII and Affects Its Multimerization

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4663-4670 ◽  
Author(s):  
S. Jorieux ◽  
C. Gaucher ◽  
J. Goudemand ◽  
C. Mazurier
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Stop Codon ◽  
Novel Mutation ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequencing Analysis ◽  
Normal Platelet ◽  
Von Willebrand ◽  
Willebrand Factor

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.

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