MONOCLONAL ANTIBODY WITH PREFERENTIAL AFFINITY FOR LARGE MULTIMERS OF VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
Tomiko Ryu ◽  
Akiko Nakayama ◽  
Atsushi Oguchi ◽  
Tadatoshi Kinoshita ◽  
Mutsuyoshi Kazama ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Gel Filtration ◽  
Void Volume ◽  
Size Fractions ◽  
Large Size ◽  
Function Relationship ◽  
Von Willebrand ◽  
Relationship Of ◽  
Willebrand Factor

von Willebrand factor (vWF) consists of a series of multimers of 270,000 mol. wt. subunits. Ristocetin cofactor activity (vWF:RCo) and capacity of binding to platelets of vWF are associated with large multimers, and Type IIA von Willebrand's disease (vWD) is characterized by lack of the large multimers. The significance of multimeric structure in relation to vWF function remains unclear. We obtained a monoclonal antibody (MAb) to human vWF which inhibited ristocetin-induced platelet aggregation. This antibody proved to bind preferentially to larger multimers by the finding that the MAb-conjugated Sepharose adsorbed large multimers of vWF from cryoprecipitates, leaving small multimers unadsorbed. vWF:Ag levels of noraml subjects determined by ELISA using the MAb correlated well with those by ELISA using polyclonal antibody (PAb) to vWF. When the plasma from patients with Type IIA vWD and platelet type vWD were examined, the values obtained by the MAb ELISA had a good correlation with vWF:RCo, but were lower than the values obtained by the PAb ELISA. In gel filtration of factor VIII concentrate, vWF:Ag detected by the MAb and vWF:RCo were present in the void volume and large size fractions, whereas vWF:Ag detectable with PAb appeared broadly from the void volume to smaller size fractions. The MAb inhibited ristocetin-dependent binding of vWF to platelets, but did not affect ADP-induced binding of vWF to platelets. These findings suggest that the large multimers have a function-associated specific structure which is absent in the small multimers, and the MAb will be useful for the investigation of multimer-function relationship of vWF.

Mouse models to study von Willebrand factor structure-function relationships in vivo

2009 ◽  
Vol 29 (01) ◽  
pp. 17-20 ◽  
Author(s):  
I. Marx ◽  
I. Badirou ◽  
R. Pendu ◽  
O. Christophe ◽  
C. V. Denis
Keyword(s):  
Structure Function ◽  
Transient Expression ◽  
Von Willebrand Factor ◽  
Large Size ◽  
Mouse Hepatocytes ◽  
Function Relationship ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.

Thrombin Binding To Factor VIII/Von Willebrand Factor Protein

1981 ◽  
Author(s):  
M E P Switzer ◽  
P A McKee
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Substrate Binding ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Void Volume ◽  
Peptidase Activity ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

Thrombin (IIa) both activates and inactivates the procoagulant activity of Factor VIII/von Willebrand Factor (FVIII/vWF). The level of activation increases as the IIa: FVIII/vWF ratio approaches 1:1, suggesting that IIa might bind stoichiometrically to FVIII/vWF either during or after activation. We approached this question by gel filtration and ultracentrifugation studies of FVIII/vWF and l25I-IIa, which activated FVIII/vWF as well as unlabeled IIa. When the mixture of 125I-IIa and FVIII/vWF was chromatographed on 4% agarose a peak of 125I-IIa was eluted with the FVIII/ vWF in the void volume (V0). Similarly, when 125I-IIa was ultracentrifuged with FVIII/vWF, a peak of radioactivity sedimented with the FVIII/vWF protein. 125I-aibumin, used to approximate a control, did not bind to FVIII/vWF. The 125I-IIa-FVIII/vWF complex isolated from the 4% agarose filtration retained ∼50% peptidase activity. The ability to activate additional FVIII/vWF or to clot fibrinogen was <10% of that of free IIa isolated from the same chromatogram. Both the FVIII and vWF moieties appear to be important in binding, since VD protein isolated from the gel filtration of FVIII/vWF on 4% agarose in 0.25 M CaCl2 binds about 24% as much 125I-IIa as native FVIII/vWF. When the isolated 125I-IIa-FVIII/vWF complex was rechromatographed on 4% agarose in 0.15 M NaCl, essentially no dissociation occurred. When these experiments were repeated in 4 M guanidine hydrochloride (GnHCl), ∼30% of the IIa remained bound. When the 125I-IIa-FVIII/vWF complex was isolated from the GnHCl chromatography and analyzed by SDS-PAGE, 58% of the IIa remained bound to the FVIII/vWF before reduction and 43% of the IIa remained bound even after reduction with β-mercaptoethanol for 3 hours at 37°. Thus FVIII/vWF binds at least some of the IIa very tightly. Since FVIII/vWF-bound thrombin is essentially inactive toward macromolecular substrate, binding of thrombin to FVIII/vWF is most likely a mechanism for removing active thrombin from the circulation.

scholarly journals Structure-function relationship of human von Willebrand factor

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 605-611 ◽  
Author(s):  
JP Girma ◽  
D Meyer ◽  
CL Verweij ◽  
H Pannekoek ◽  
JJ Sixma
Keyword(s):  
Structure Function ◽  
Von Willebrand Factor ◽  
Structure Function Relationship ◽  
Function Relationship ◽  
Von Willebrand ◽  
Relationship Of ◽  
Willebrand Factor

Characterisation of a Monoclonal Antibody to von Willebrand Factor as a Potent Inhibitor of Ristocetin-Mediated Platelet Interaction and Platelet Adhesion

1987 ◽  
Vol 57 (03) ◽  
pp. 278-282 ◽  
Author(s):  
Sylvie Jorieux ◽  
Christophe de Romeuf ◽  
Bruno Samor ◽  
Maurice Goudemand ◽  
Claudine Mazurier
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Potent Inhibitor ◽  
Disease Patient ◽  
Structure Relationship ◽  
Von Willebrand ◽  
Relationship Of ◽  
Willebrand Factor ◽  
Platelet Functions

SummaryWe studied a murine monoclonal antibody (211 A6) to von Willebrand factor (vWF) with a view to investigating structure-relationship. of plasma vWF. The specificity of this antibody has been substantiated by ELISA tests and indirect immunofluorescence. It reacts with purified vWF, normal plasma but not with plasma or platelets from a severe von Willebrand’s disease patient. Monoclonal antibody 211 A6 is a potent inhibitor of ristocetin-induced platelet aggregation. The 125I-FVIII/vWF binding to platelets in presence of ristocetin is totally inhibited by low 211 A6 concentrations. Thrombin-induced binding of vWF to platelets is not affected by 211 A6. The ability of this antibody to inhibit platelet adhesion to subendothelium and to collagen was investigated with a perfusion model. The complete inhibition of platelet adhesion by 211 A6 questions the similarity or the interrelationship in vWF domains involved in ristocetin-induced platelet functions and platelet adhesion.

scholarly journals Structure-function relationship of human von Willebrand factor

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 605-611
Author(s):  
JP Girma ◽  
D Meyer ◽  
CL Verweij ◽  
H Pannekoek ◽  
JJ Sixma
Keyword(s):  
Structure Function ◽  
Von Willebrand Factor ◽  
Structure Function Relationship ◽  
Function Relationship ◽  
Von Willebrand ◽  
Relationship Of ◽  
Willebrand Factor

Porcine von Willebrand Factor Binding to Human Platelet GPIb Induces Transmembrane Calcium Influx

1996 ◽  
Vol 75 (04) ◽  
pp. 655-660 ◽  
Author(s):  
Mario Mazzucato ◽  
Luigi De Marco ◽  
Paola Pradella ◽  
Adriana Masotti ◽  
Francesco I Pareti
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Transmembrane Flux ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryPorcine von Willebrand factor (P-vWF) binds to human platelet glycoprotein (GP) lb and, upon stirring (1500 rpm/min) at 37° C, induces, in a dose-dependent manner, a transmembrane flux of Ca2+ ions and platelet aggregation with an increase in their intracellular concentration. The inhibition of P-vWF binding to GP lb, obtained with anti GP lb monoclonal antibody (LJ-Ib1), inhibits the increase of intracellular Ca2+ concentration ([Ca2+]i) and platelet aggregation. This effect is not observed with LJ-Ib10, an anti GP lb monoclonal antibody which does not inhibit the vWF binding to GP lb. An anti GP Ilb-IIIa monoclonal antibody (LJ-CP8) shown to inhibit the binding of both vWF and fibrinogen to the GP IIb-IIIa complex, had only a slight effect on the [Ca2+]i rise elicited by the addition of P-vWF. No inhibition was also observed with a different anti GP IIb-IIIa monoclonal antibody (LJ-P5), shown to block the binding of vWF and not that of fibrinogen to the GP IIb-IIIa complex. PGE1, apyrase and indomethacin show a minimal effect on [Ca2+]i rise, while EGTA completely blocks it. The GP lb occupancy by recombinant vWF fragment rvWF445-733 completely inhibits the increase of [Ca2+]i and large aggregates formation. Our results suggest that, in analogy to what is seen with human vWF under high shear stress, the binding of P-vWF to platelet GP lb, at low shear stress and through the formation of aggregates of an appropriate size, induces a transmembrane flux of Ca2+, independently from platelet cyclooxy-genase metabolism, perhaps through a receptor dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of the GP IIb-IIIa complex.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

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