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

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.

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.

scholarly journals Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2335-2344 ◽  
Author(s):  
Christelle Perrault ◽  
Hanneke Lankhof ◽  
Dominique Pidard ◽  
Danièle Kerbiriou-Nabias ◽  
Jan J. Sixma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Binding Domain ◽  
Rgd Sequence ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Endothelial Cell Adhesion

Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

scholarly journals Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2335-2344 ◽  
Author(s):  
Christelle Perrault ◽  
Hanneke Lankhof ◽  
Dominique Pidard ◽  
Danièle Kerbiriou-Nabias ◽  
Jan J. Sixma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Binding Domain ◽  
Rgd Sequence ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Endothelial Cell Adhesion

AbstractEndothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

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.

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.

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.

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.

Glycoprotein Ib Can Mediate Endothelial Cell Attachment to a von Willebrand Factor Substratum

1995 ◽  
Vol 73 (02) ◽  
pp. 309-317 ◽  
Author(s):  
Dorothy A Beacham ◽  
Miguel A Cruz ◽  
Robert I Handin
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Single Amino Acid ◽  
Cell Surface Expression ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryIntroduction of single amino acid substitutions into the C-terminal Arg-Gly-Asp-Ser (RGDS) site of von Willebrand Factor, referred to as RGD mutant vWF, selectively abrogated vWF binding to platelet glycoprotein IIb/IIIa (GpIIb/IIIa, αIIbβ3 and abolished human umbilical vein endothelial cell (HUVEC) spreading, but not attachment, to RGD mutant vWF (Beacham, D. A., Wise, R. J., Turci, S. M. and Handin, R. I. 1992. J. Biol. Chem. 167, 3409-3415). These results suggested that in addition to the vitronectin receptor (VNR, αvβ3), a second endothelial membrane glycoprotein can mediate HUVEC adhesion to vWF. HUVEC attachment to wild-type (WT) and RGD-mutant vWF was reduced by two proteins known to block the vWF-platelet glycoprotein Ib/IX (GpIb/IX) interaction, the monoclonal antibody AS-7 and the recombinant polypeptide, vWF-A1. The addition of cytochalasin B or DNase I to disrupt potential GPIbα-cytoskeletal interactions enhanced the immunoprecipitation of endothelial GPIbα, caused HUVEC to round up, and increased HUVEC adhesion to RGD mutant vWF. These results indicate that while the VNR is the primary adhesion receptor for vWF, endothelial GPIbα can mediate HUVEC attachment to vWF. GpIb-dependent attachment could contribute to HUVEC adhesion under conditions when cell surface expression of the VNR is downregulated, and VNR-dependent adhesion is reduced.

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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