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.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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 Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2048-2055 ◽  
Author(s):  
PA Kroner ◽  
ML Kluessendorf ◽  
JP Scott ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

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.

scholarly journals Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2788-2794 ◽  
Author(s):  
T Englender ◽  
A Lattuada ◽  
PM Mannucci ◽  
JE Sadler ◽  
A Inbal
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Patient Characteristics ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Disease Mutations ◽  
Normal Transport ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Intracellular Proteolysis

Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90- 2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation.

The arginine-552-cysteine (R1315C) mutation within the A1 loop of von Willebrand factor induces an abnormal folding with a loss of function resulting in type 2A–like phenotype of von Willebrand disease: study of 10 patients and mutated recombinant von Willebrand factor

Blood ◽  
2001 ◽  
Vol 97 (4) ◽  
pp. 952-959 ◽  
Author(s):  
Anne-Sophie Ribba ◽  
Lysiane Hilbert ◽  
Jean-Maurice Lavergne ◽  
Edith Fressinaud ◽  
Catherine Boyer-Neumann ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
In Vitro Mutagenesis ◽  
Loss Of Function ◽  
Prolonged Bleeding Time ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The study identified 10 patients from 6 families with prolonged bleeding time, decreased von Willebrand factor (vWF) ristocetin cofactor activity (RCoF) to vWF:Ag (antigen) ratio, and reduced ristocetin-induced platelet agglutination as well as ristocetin- or botrocetin-induced binding of plasma vWF to platelet glycoprotein Ib (GpIb). In addition, all patients showed a decrease of intermediate-molecular-weight (intermediate-MW) and high-molecular-weight (HMW) multimers of vWF. In the heterozygous state, a cysteine-to-threonine (C → T) transversion was detected at nucleotide 4193 of the VWF gene of all patients and lead to the arginine (R)522C substitution in the A1 loop of vWF mature subunit (R1315C in the preprovWF). By in vitro mutagenesis of full-length complementary DNA (cDNA) of vWF and transient expression in COS-7 cells, the mutated C552 recombinant vWF (C552rvWF) was found to exhibit decreased expression, abnormal folding, and lack of intermediate-MW and HMW multimers. In addition, direct binding of botrocetin to C552rvWF, as well as ristocetin- and botrocetin-induced binding of C552rvWF to GpIb, was markedly decreased. Although being localized in an area of the A1 loop of vWF where most of the type 2B mutations that induce a gain-of-function have been identified, the R552C mutation induces a 2A-like phenotype with a decrease of intermediate-MW and HMW multimers as well as a loss-of-function of vWF in the presence of either ristocetin or botrocetin.

scholarly journals Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2048-2055 ◽  
Author(s):  
PA Kroner ◽  
ML Kluessendorf ◽  
JP Scott ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

Diagnosis of Subtype 2B von Willebrand Disease in a Patient with 2A Phenotype of Plasma von Willebrand Factor

1995 ◽  
Vol 73 (04) ◽  
pp. 610-616 ◽  
Author(s):  
Christine Gaucher ◽  
Christophe de Romeuf ◽  
Michéle Rauïs-Morret ◽  
Francis Corazza ◽  
Pierre Fondu ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Correct Diagnosis ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Diagnosis And Classification ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2A of von Willebrand disease refers to qualitative variants with decreased platelet dependent function that is associated with the absence of high molecular weight forms of von Willebrand factor (vWF) multimers. Type 2B refers to qualitative variants with increased affinity for platelet glycoprotein lb. In this report we describe the study of a patient who has been previously diagnosed as having subtype 2A von Willebrand disease (vWD), because she had no heightened ristocetin-induced platelet aggregation, no large and intermediate molecular weight von Willebrand factor (vWF) multimers in plasma, and no increase in plasma vWF capacity to bind to normal platelets in the presence of low ristocetin concentrations. The DNA sequencing of the 3’ part of the exon 28 of the vWF gene where most of the subtype 2A mutations have already been identified, did not detect any nucleotide change. At variance, a G to A transition changing the encoded amino acid residue from Val 553 to Met in mature vWF, was found in the 5’ part of this exon. This mutation which has already been found in several unrelated families with 2B vWD and the increased binding of the patient platelet vWF on normal platelets in the presence of low ristocetin concentrations provide evidence for subtype 2B vWD. This study thus illustrates the importance of the molecular characterization of patients in the correct diagnosis and classification of type 2 vWD.

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.

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.

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