Distinct abnormalities in the interaction of purified types IIA and IIB von Willebrand factor with the two platelet binding sites, glycoprotein complexes Ib-IX and IIb-IIIa.

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

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Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin

Blood ◽

10.1182/blood.v64.4.797.bloodjournal644797 ◽

1984 ◽

Vol 64 (4) ◽

pp. 797-800 ◽

Cited By ~ 4

Author(s):

HR Gralnick ◽

SB Williams ◽

BS Coller

Keyword(s):

Monoclonal Antibodies ◽

Von Willebrand Factor ◽

Adenosine Diphosphate ◽

Glycoprotein Ib ◽

Human Fibrinogen ◽

Factor Binding ◽

Normal Plasma ◽

Platelet Binding ◽

Von Willebrand ◽

Willebrand Factor

Two monoclonal antibodies--one that blocks ristocetin-induced platelet binding of von Willebrand factor to glycoprotein Ib and one that blocks adenosine diphosphate-induced binding of fibrinogen to the glycoprotein IIb/IIIa complex--were used to assess the binding site(s) for von Willebrand factor when platelets are stimulated with thrombin or adenosine diphosphate (ADP). Neither agonist induced binding of von Willebrand factor to glycoprotein Ib. ADP and thrombin induced von Willebrand factor binding exclusively to the glycoprotein IIb/IIIa complex. The results of the site of binding of von Willebrand factor with thrombasthenic platelets were consistent with the data obtained with the monoclonal antibodies and normal platelets. Human fibrinogen caused complete inhibition of thrombin-induced von Willebrand factor binding to normal platelets at concentrations considerably below that found in normal plasma. We conclude that thrombin induces very little binding of exogenous von Willebrand factor to platelets at normal plasma fibrinogen levels.

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The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets

10.1055/s-0039-1687415 ◽

1979 ◽

Author(s):

H.R. Gralnick ◽

D.K. Morisato

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Binding Sites ◽

Human Platelets ◽

Galactose Oxidase ◽

Scatchard Analysis ◽

Human Fibrinogen ◽

Number Of Binding Sites ◽

Von Willebrand ◽

Willebrand Factor

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.

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Novel aptamer to von Willebrand factor A1 domain (TAGX-0004) shows total inhibition of thrombus formation superior to ARC1779 and comparable to caplacizumab

Haematologica ◽

10.3324/haematol.2019.235549 ◽

2019 ◽

Vol 105 (11) ◽

pp. 2631-2638 ◽

Cited By ~ 4

Author(s):

Kazuya Sakai ◽

Tatsuhiko Someya ◽

Kaori Harada ◽

Hideo Yagi ◽

Taei Matsui ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Von Willebrand Factor ◽

Plasmon Resonance ◽

Binding Sites ◽

Thrombus Formation ◽

Static Conditions ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction between its A1 domain and platelet glycoprotein Ib. ARC1779, an aptamer to the VWF A1 domain, was evaluated in a clinical trial for acquired thrombotic thrombocytopenic purpura (aTTP). Subsequently, caplacizumab, an anti-VWF A1 domain nanobody, was approved for aTTP in Europe and the United States. We recently developed a novel DNA aptamer, TAGX-0004, to the VWF A1 domain; it contains an artificial base and demonstrates high affinity for VWF. To compare the effects of these three agents on VWF A1, their ability to inhibit ristocetin- or botrocetin-induced platelet aggregation under static conditions was analyzed, and the inhibition of thrombus formation under high shear stress was investigated in a microchip flow chamber system. In both assays, TAGX-0004 showed stronger inhibition than ARC1779, and had comparable inhibitory effects to caplacizumab. The binding sites of TAGX-0004 and ARC1779 were analyzed with surface plasmon resonance performed using alanine scanning mutagenesis of the VWF A1 domain. An electrophoretic mobility shift assay showed that R1395 and R1399 in the A1 domain bound to both aptamers. R1287, K1362, and R1392 contributed to ARC1779 binding, and F1366 was essential for TAGX-0004 binding. Surface plasmon resonance analysis of the binding sites of caplacizumab identified five amino acids in the VWF A1 domain (K1362, R1392, R1395, R1399, and K1406). These results suggested that TAGX-0004 possessed better pharmacological properties than caplacizumab in vitro and might be similarly promising for aTTP treatment.

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Comparison of von Willebrand factor platelet‐binding activity assays: ELISA overreads type 2B with loss of HMW multimers

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.14971 ◽

2020 ◽

Vol 18 (10) ◽

pp. 2513-2523

Author(s):

Attila Szederjesi ◽

Luciano Baronciani ◽

Ulrich Budde ◽

Giancarlo Castaman ◽

Paola Colpani ◽

...

Keyword(s):

Von Willebrand Factor ◽

Binding Activity ◽

Platelet Binding ◽

Von Willebrand ◽

Willebrand Factor

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Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor

Blood ◽

10.1182/blood.v88.10.3824.bloodjournal88103824 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3824-3830 ◽

Cited By ~ 25

Author(s):

TH Mondoro ◽

CD Wall ◽

MM White ◽

LK Jennings

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Binding Sites ◽

Flow Cytometric Analysis ◽

Clot Retraction ◽

Recognition Sequence ◽

Ligand Interaction ◽

Selective Ligand ◽

Von Willebrand ◽

Willebrand Factor

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex- activating antibody induces fibrinogen-and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD- containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand- receptor interactions that directly mediate platelet aggregation.

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The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

10.1055/s-0038-1652985 ◽

1981 ◽

Author(s):

P Silber ◽

T H Finlay

Keyword(s):

Von Willebrand Factor ◽

Binding Sites ◽

Binding Constant ◽

Specific Binding ◽

Human Platelets ◽

Scatchard Analysis ◽

Binding Data ◽

Number Of Binding Sites ◽

Von Willebrand ◽

Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

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