Correspondence to: Xiaoping Du, Department of Pharmacology, the University of Illinois, College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612. Tel:(312) 355-0237 Fax:(312) 996-1225 E-mail:xdu{at}uic.edu.
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Abstract |
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We have reconstituted the platelet glycoprotein (GP) Ib-IXmediated activation of the integrin IIbß3 in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing
IIbß3 adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin
IIbß3 and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against
IIbß3. vWF binding to GPIb-IX also activates soluble fibrinogen binding to
IIbß3 indicating that GPIb-IX mediates a cellular signal leading to
IIbß3 activation. Deletion of the 14-3-3binding site in GPIb
inhibited GPIb-IXmediated fibrinogen binding to
IIbß3 and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIb
induced an endogenous integrin-dependent cell spreading on vWF without requiring
IIbß3, but inhibited vWF-induced fibrinogen binding to
IIbß3. Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IXmediated activation of
IIbß3 requires 14-3-3 interaction with GPIb
.
Key Words: platelet, glycoprotein Ib-IX, integrin, 14-3-3, von Willebrand factor
PLATELET adhesion to the subendothelial matrix plays a critical role in thrombosis and hemostasis. Initial platelet adhesion is mediated by the interaction between a platelet receptor for von Willebrand factor (vWF), the glycoprotein Ib-IX complex (GPIb-IX), and matrix-bound vWF (IIbß3 interaction with the RGD sequence in vWF, and platelet aggregation is dependent upon activation of integrin
IIbß3 binding to soluble fibrinogen (for reviews see
GPIb-IX consists of three subunits: GPIb, GPIbß, and GPIX. GPIb-IX is loosely associated with glycoprotein V. The NH2-terminal domain of GPIb
contains binding sites for vWF and thrombin (for reviews see
(
(
contains a binding site for filamin (also called actin-binding protein or ABP-280), which links GPIb-IX to cross-linked actin filamental structures underlining the plasma membrane (the membrane skeleton;
, is also associated with GPIb-IX (
is located in a 15amino acid residue region (residues 595610) at the COOH terminus of GPIb
(
is regulated by phosphorylation of GPIb
at serine609 (
(
binding requires the helix I region of 14-3-3
distinct from the site required for binding of RSXpSXP-containing ligands (
In this study, we have established a CHO cell expression model for studying GPIb-IXmediated integrin activation. We show that deletion of the 14-3-3binding site in the COOH terminus of GPIb inhibits GPIb-IXinduced integrin activation. Further, we show that expression of a dominant negative mutant of 14-3-3 containing the GPIb-IXbinding site also inhibits vWF induced integrin activation. Thus, 14-3-3 plays important roles in vWF-induced GPIb-IX signaling leading to
IIbß3 activation. In addition, deleting both the filamin and 14-3-3binding sites in GPIb
enhanced cell spreading on vWF. This did not require
IIbß3, but did require an endogenous integrin. The same deletion mutant, however, failed to mediate vWF-induced fibrinogen binding, suggesting that
IIbß3 activation was inhibited. Thus, different mechanisms may be responsible for vWF interaction with different integrins after GPIb-IXmediated initial adhesion.
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Materials and Methods |
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Reagents
An anti-peptide antibody, anti-IbC, recognizing the COOH-terminal domain of GPIb
has been described previously (
, and purification of vWF and botrocetin were as described previously (
, P3221, was kindly provided by Dr. Zaverio Ruggeri (The Scripps Research Institute, La Jolla, CA). mAbs D57 and 15 against integrin
IIbß3 were kindly provided by Dr. Mark Ginsberg (The Scripps Research Institute, La Jolla, CA). Monoclonal antibody, 4F10, against integrin
IIbß3 complex was kindly provided by Dr. Virgil Woods (University of California at San Diego, CA). Monoclonal antibody against GPIb
, SZ2, monoclonal antibody against human ß3, SZ21, and monoclonal antibody against vWF, SZ29, were generous gifts from Dr. Changgeng Ruan (Suzhou Medical College, Suzhou, China;
IIb and ß3 in CDM8 vector were kindly provided by Dr. Mark Ginsberg. In some experiments, botrocetin was also purchased from Centerchem. Ristocetin was purchased from Sigma Chemical Co.
DNA encoding wild-type and mutant 14-3-3 was described previously (
were subcloned into pEGFP-C2 vector (Clonetech) between EcoRI and XbaI sites. The constructs encode a wild-type or a mutant 14-3-3
fused to the COOH terminus of green fluorescent protein (GFP)1.
Cell Lines Expressing Recombinant Proteins
Transfections of cDNA into CHO cells were performed according to the previously described methods using Lipofectamine (BRL; (P3221) and/or integrin
IIbß3 (D57). The following cell lines were established: cells expressing GPIb-IX complex (1b9) (
IIbß3 (2b3a); cells coexpressing GPIb-IX and
IIbß3 (123); and cells coexpressing integrin
IIbß3 and GPIb-IX mutants with truncated GPIb
cytoplasmic domains at residues 591 (
591/2b3a) and 559 (
559/2b3a) (
Cell Adhesion Assay
Microtiter wells were coated with 10 µg/ml vWF or fibrinogen in PBS at 4°C overnight. Cells in Tyrode's buffer in the presence of 5 µg/ml botrocetin were incubated in ligand-coated microtiter wells for 30 min at 37°C in a CO2 incubator. As adhesion of the GPIb-IX and integrin-transfected CHO cells to vWF does not require botrocetin, botrocetin was omitted in some experiments. After three washes, cell spreading was examined under an inverted microscope (20x objective lens). In quantitative assays, 50 µl of 0.3% p-nitrophenyl phosphate in 1% Triton X-100, 50 mM sodium acetate, pH 5.0, was added to microtiter wells and incubated at 37°C for 1 h. The reaction was stopped by adding 50 µl of 1 M NaOH. Results were determined by reading OD at 405 nm wave length. A standard curve of acid phosphatase reaction was established by adding the acid phosphatase substrate to various known numbers of the same cells in parallel wells. Acid phosphatase assay of the standards confirmed that the OD value was proportional to cell number. The rate of cell adhesion was estimated from the ratio of the numbers of adherent cells to that of total cells.
Fluorescence Microscopy
Cells were allowed to adhere and spread on vWF- or fibrinogen-coated glass chamber slides (Nunc). After three washes, cells were fixed by adding 4% paraformaldehyde in PBS. In experiments that required cell permeabilization, cells were permeabilized by adding 0.1 M Tris, 0.01 M EGTA, 0.15 M NaCl, 5 mM MgCl2, pH 7.4, containing 0.1% Triton X-100, 0.5 mM leupeptin, 1 mM PMSF, and 0.1 mM E64. The cells were then incubated with 20 µg/ml of various antibodies at 22°C for 1 h. After three washes, cells were further incubated with fluorescein- or rhodamine-labeled secondary antibodies at 22°C for 30 min. To stain the actin filaments, rhodamine-labeled phalloidin (Sigma Chemical Co.) was also added. After additional washes, cells were photographed under a fluorescence microscope. In some experiments, the data were collected by a cooled CCD camera and surface area quantitated using Image-Pro Plus (Media Cybernetics).
Flow Cytometry Analysis of vWF Binding and vWF-induced Fibrinogen Binding
Fluorescein-labeling of fibrinogen was prepared as described previously (1 x 107/ml) were incubated for 30 min with 15 µg/ml fluorescein-labeled fibrinogen in the presence of 20 µg/ml vWF and 1 mg/ml ristocetin. As a negative control, cells were also incubated with fluorescein-labeled fibrinogen in the presence of 1 mg/ml ristocectin but in the absence of vWF. RGDS peptide (1 mM) was added in parallel assays for estimation of specific fibrinogen binding to the integrin. We showed previously that 1 mM RGDS completely abolished fibrinogen binding to integrin
IIbß3 while 1 mM RGES had no effect (
For vWF binding, the cells in Tyrode's buffer were incubated for 30 min at 22°C with vWF in the presence 1 mg/ml ristocetin. After washing, the cells were further incubated for 30 min with a monoclonal antibody against vWF, SZ29, and then analyzed by flow cytometry.
Immunoprecipitation
CHO cells coexpressing integrin IIbß3 with wild-type GPIb-IX (123 cells) or GPIb-IX mutant
591 were solubilized as previously described (
or mouse IgG (Sigma Chemical Co.) at 4°C for 1 h and further incubated for 1 h after addition of protein Gconjugated Sepharose beads (Sigma Chemical Co.). After three washes, the bead-bound proteins were analyzed by SDS-PAGE and Western blotting with a rabbit anti-GPIb
antibody or a rabbit antibody against 14-3-3
(
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Results |
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Roles of GPIb-IX and IIbß3 in Mediating Cell Adhesion to vWF
To analyze the roles of GPIb-IX and integrin IIbß3 in vWF-mediated platelet adhesion and activation, stable CHO cell lines were established that express one of the two platelet receptors for vWF: GPIb-IX (1b9 cells) or integrin
IIbß3 (2b3a cells). A stable cell line was also established that expressed both GPIb-IX and integrin
IIbß3 at levels comparable to 1b9 and 2b3a cells, respectively (123 cells; Fig 1 A). These cells were incubated in vWF-coated microtiter wells for 30 min in the presence of botrocetin, which binds to vWF and mimics the effects of subendothelial matrix to induce vWF binding to GPIb-IX (
IIbß3) adhered to the vWF-coated surface compared with
55% adhesion to fibrinogen, suggesting only a background level of vWF-integrin interaction. This result is consistent with previous work showing a low affinity state of
IIbß3 expressed in CHO cells (
IIbß3 interacts poorly with vWF without prior activation (
IIbß3 without prior activation (
IIbß3.
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In contrast to cells expressing IIbß3 (2b3a cells), those expressing GPIb-IX (1b9 cells) or those expressing both GPIb-IX and integrin
IIbß3 (123 cells), adhered to vWF-coated wells in the presence of botrocetin (Fig 1 B). As platelet adhesion to immobilized vWF occurs in the absence of vWF modulators (
IIbß3 to vWF is similar to platelet adhesion. Furthermore, adhesion of 123 cells to vWF was inhibited by monoclonal antibodies against vWF-binding site of GPIb
(Fig 1 C). These results suggest that, as in platelets, GPIb-IX is required for cell adhesion to vWF in this CHO cell expression model.
GPIb-IX Induces Integrin-vWF Interaction and Integrin-dependent Cell Spreading on vWF
Under a microscope, most adherent 1b9 cells (expressing GPIb-IX only) on vWF showed a rounded morphology similar to nonadherent cells (Fig 2 A). In contrast, 123 cells (coexpressing GPIb-IX and IIbß3) spread on the vWF-coated surface (Fig 2 A). Spreading of 123 cells was abolished by RGDS peptide (Fig 2 A), indicating that spreading was mediated by integrins. Spreading of 123 cells was also inhibited by the monoclonal antibody 4F10, against human
IIbß3 complex, and by anti-human ß3 antibody SZ21 (Fig 2 A). These data indicated that spreading was mainly mediated by integrin
IIbß3 and that endogenous integrins were unlikely to play a major role. It is unlikely that coexpression of GPIb-IX with
IIbß3 in the 123 cell line resulted in constitutively active integrin
IIbß3, as 123 cells did not bind to soluble fibrinogen without prior activation (data not shown, see Fig 3). Thus, vWF binding to GPIb-IX induces integrin-vWF interaction and integrin-mediated cell spreading. To examine whether GPIb-IXmediated signaling pathway in CHO cells mimics that in platelets, we examined the effects of platelet activation inhibitors. We found that the PGE1, which elevates intracellular cAMP, wortmannin, and calphostin C, which inhibit PI-3 kinase and PKC, respectively, also inhibited GPIb-IX and integrin-dependent CHO cell spreading on vWF. Thus, GPIb-IX expressed in CHO cells induced integrin interaction with vWF in a manner similar to that in platelets.
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To further exclude the possibility that integrin function in 123 cells may differ from that in the 2b3a cell line, we also examined integrin-mediated cell spreading on immobilized fibrinogen. Both 2b3a cells and 123 cells fully spread on immobilized fibrinogen (Fig 2 B), suggesting that IIbß3 expressed in both cell lines functioned in a similar manner. As shown above, only a small percentage of 2b3a cells adhere to vWF. Some of these adherent cells, however, also spread on vWF, suggesting that the background level GPIb-IXindependent interaction of
IIbß3 with vWF in a small percentage of 2b3a cells can also mediate cell spreading.
To examine the morphological changes in more detail, the adherent cells were stained with fluorescently labeled phalloidin, and examined by fluorescence microscopy under high magnification. Only 5% of 1b9 cells spread on vWF (Fig 2 C). Most 1b9 cells did not spread or only poorly spread on vWF. However, 58% of these poorly spread cells showed limited filopodium- or lamellipodium-like structures extending to the vWF-coated surface (Fig 2 C) which was inhibited by RGDS peptide. This indicates a low level interaction between vWF and an endogenous integrin, which is consistent with the results obtained by 70% of 123 cells (expressing both GPIb-IX and integrin
IIbß3) fully spread, which was inhibited by RGDS peptide (Fig 2 C). These results show that GPIb-IX induces integrin
IIbß3 interaction with vWF which is responsible for 123 cell spreading on vWF.
vWF-induced Fibrinogen Binding to Integrin IIbß3 in CHO Cells
Two possible mechanisms could explain GPIb-IXinduced integrin-vWF interaction in the CHO cell expression model: (a) GPIb-IX may induce a cellular signal that increases the affinity of integrin for vWF (activation); or (b) the GPIb-IX binding to vWF may allow access of integrin to vWF, e.g., by changing the conformation of vWF. To differentiate between these two possibilities, we examined whether vWF activated integrin binding to another ligand of IIbß3, soluble fibrinogen, in 123 cells. It is known that integrin
IIbß3 binds soluble fibrinogen only after the integrin is activated (for reviews, see
IIbß3 (Fig 3 A). When both vWF and ristocetin were present, however, there was significant binding of fibrinogen. vWF-induced fibrinogen binding was inhibited by RGDS peptide (Fig 3B and Fig E), and was also inhibited by an anti-GPIb
monoclonal antibody, AK2, known to inhibit ristocetin-induced vWF binding to GPIb-IX (Fig 3 C). Furthermore, vWF did not induce specific fibrinogen binding to 2b3a cells (Fig 3 D), suggesting that vWF-induced fibrinogen binding to integrin
IIbß3 requires vWF interaction with GPIb-IX. Thus, vWF interaction with GPIb-IX not only stimulates vWF-
IIbß3 interaction, but also induces the integrin to bind soluble fibrinogen. These data indicate that ristocetin-dependent vWF binding to GPIb-IX induces a cellular signal that activates the ligand-binding function of
IIbß3.
Effects of GPIb Cytoplasmic Domain Deletion Mutagenesis on 14-3-3binding Function of GPIb-IX
We showed previously ( binds to a site in the COOH-terminal 15 residues (residues 595610) of the cytoplasmic domain of GPIb
. To investigate the role of 14-3-3 in GPIb-IXmediated activation of
IIbß3 in the CHO cell model, we established a CHO cell line (
591/2b3a cells) that coexpresses
IIbß3 and a mutant GPIb-IX,
591, that lacks the 14-3-3binding site (18 residues) at the COOH terminus of GPIb
, but retains the functional filamin-binding domain in GPIb
(
antibodies (Fig 4). The mutant GPIb-IX (
591), however, failed to coimmunoprecipitate endogenous CHO cell 14-3-3 (Fig 4). As a control, we also immunoblotted the same immunoprecipitates with an anti-GPIb
antibody, and observed that similar amounts of GPIb
were immunoprecipitated from both the
591/2b3a cells and 123 cells (expressing wild-type GPIb-IX; Fig 4). Thus, the
591 mutant GPIb-IX is defective in binding to endogenous 14-3-3.
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GPIb-IXmediated Activation of the Integrin IIbß3 Requires the 14-3-3binding Site in GPIb
To determine whether deletion of the 14-3-3binding site in GPIb affects GPIb-IXmediated activation of the integrin
IIbß3, we examined whether ristocetin-induced vWF binding to the mutant GPIb-IX stimulates the binding of FITC-labeled fibrinogen to
591/2b3a cells. Fig 5 A shows that vWF induces soluble fibrinogen binding to 123 cells which is inhibited by RGDS peptide. In contrast, vWF-induced fibrinogen binding to
591/2b3a cells is absent. It is unlikely that the defect in fibrinogen binding to
591/2b3a cells results from naturally occurring mutations developed in the CHO cells during selection as the
591/2b3a cells are established by mass sorting of cells reactive with both antibodies against
IIbß3 and GPIb-IX and not by single cell cloning. It is also unlikely that the inhibition of integrin activation results from defective binding of vWF as vWF binding to 591/2b3a cells is not negatively affected (Fig 6). As
591/2b3a cells adhered and spread on fibrinogen (Fig 7), the possibility of a defective integrin function can be further excluded. Thus, our data indicate that the 14-3-3binding site of GPIb
plays an important role in GPIb-IXmediated integrin activation.
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We also examined vWF-induced fibrinogen binding to a CHO cell line (559/2b3a), expressing integrin
IIbß3 and a truncation mutant GPIb-IX lacking both the 14-3-3binding domain and filamin-binding domain of GPIb
. No specific fibrinogen binding was detected in this cell line suggesting that inhibition of
IIbß3 activation by deleting the 14-3-3binding site of GPIb
was not reversed by further deletion of the filamin-binding site of GPIb
(Fig 5).
The 14-3-3binding Site of GPIb-IX Is Involved in GPIb-IXinduced Integrin-vWF Interaction and Cell Spreading on vWF
To investigate whether 14-3-3 binding plays a role in GPIb-IXinduced integrin-vWF interaction and integrin-dependent cell spreading on vWF, the 123 cells and 591/2b3a cells were allowed to adhere to vWF-coated microtiter wells. As examined under the microscope,
70% of the 123 cells were spread on both vWF- and fibrinogen-coated microtiter wells. In contrast, only a small percentage (
30%) of
591/2b3a cells appeared spreading on vWF, indicating that GPIb-IXinduced integrin-vWF interaction was inhibited (Fig 7). To quantitate the cell spreading objectively, cells adherent to vWF were permeabilized and stained with rhodamine-labeled phalloidin. Fluorescently stained cells in randomly selected fields were quantitated for cell surface area using Image-Pro Plus software (Media Cybernetics). As shown in Fig 8, the average surface area of
591/2b3a cells were about half of that of 123 cells, indicating that the spreading of the mutant cell line was significantly reduced but not totally abolished. Since
591/2b3a cells adhered and spread on fibrinogen in a manner similar to 123 cells, the ligand-binding function of
IIbß3 and the integrin-mediated spreading process was not impaired in the
591/2b3a cell line. Thus, inhibition of GPIb-IX and integrin-dependent spreading on vWF in this cell line is unlikely to be caused by a defect in ligand-binding function of
IIbß3 or in the integrin's post-ligand occupancy events. These data suggest that 14-3-3
binding to the COOH-terminal region of GPIb
plays an important role in GPIb-IXmediated activation of integrin
IIbß3. Spreading of a small percentage of mutant cells reflects a background level of
IIbß3-vWF interaction or the interaction of vWF with the endogenous CHO cell integrin (see Fig 2 C).
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Effects of Disruption of GPIb-IX Interaction with Filamin on vWF Interaction with Integrins
It has been shown previously (559) of GPIb
abolishes filamin and 14-3-3 binding to GPIb-IX, and induces GPIb-IXdependent cell spreading in the absence of integrin
IIbß3 (
559 with integrin
IIbß3 (
559/2b3a). Not only did the
559/2b3a cells exhibit no defect in spreading, but they actually showed enhanced spreading on vWF compared with 123 cells (Fig 7 and Fig 8). The spreading of
559/2b3a cells was significantly inhibited by RGDS peptide but poorly inhibited by anti-
IIbß3 antibody 4F10 and anti-ß3 antibody SZ21 (Fig 7), suggesting that an endogenous integrin plays a significant role. This result is consistent with previous studies showing that CHO cells expressing the same mutant of GPIb-IX spread on vWF in the absence of integrin
IIbß3 (
559/2b3a cells was detected (see Fig 5). These results indicate that deletion of both 14-3-3 and filamin-binding sites of GPIb
inhibited GPIb-IXmediated activation of fibrinogen binding to
IIbß3, but enhanced the interaction of vWF with an endogenous integrin (which only plays a very limited role in wild-type GPIb-IXmediated cell spreading (see Fig 2 C). Thus, it appears that two different mechanisms may be involved in the vWF interaction with integrins: a GPIb-IXmediated 14-3-3-dependent mechanism that induces an activation signal leading to the activation of integrin
IIbß3, and an alternative mechanism that allows the interaction of vWF with an unidentified integrin. The latter mechanism becomes significant only when the association of GPIb-IX with the membrane skeleton structure is disrupted.
Inhibition of GPIb-IX and Integrin-dependent Cell Spreading by a 14-3-3 Fragment Containing the GPIb-IXbinding Site
We have recently shown that GPIb binds to a site in the helix I region of 14-3-3
, distinct from the sites required for 14-3-3
binding to RSXpSXP-motif containing ligands such as c-Raf (
plays a role in GPIb-IX signaling, we constructed cDNAs encoding fusion proteins of green fluorescent protein (GFP) with wild-type 14-3-3
(GFP-1433) as well as a small fragment of 14-3-3
containing the GPIb
-binding site (1433T12, residues 188231;
70% of 123 cells adhere and spread on vWF. Cells expressing GFP-1433 fusion protein showed an increase in the percentage of spreading (85%), suggesting that overexpression of 14-3-3 enhanced cell spreading on vWF-coated surface (Fig 9). In contrast, 90% of the cells expressing GFP-1433T12 fusion protein are rounded (Fig 9), and the rest (10% cells) only partially spread on vWF (not shown). These results suggest that the small fragment of 14-3-3
inhibited the function of endogenous 14-3-3 in a dominant negative fashion.
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Discussion |
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In this study, we show that GPIb-IX binding to vWF induces signals that activate the ligand-binding function of integrin IIbß3 and integrin-dependent cell spreading using a reconstituted CHO cell expression model. We show that vWF-induced GPIb-IX signaling is inhibited by deletion of the 14-3-3binding sites in the cytoplasmic domain of GPIb
(Fig 5 and Fig 7). Thus, our study indicates that interaction between GPIb-IX and 14-3-3 plays an important role in GPIb-IXmediated signaling leading to activation of integrin
IIbß3.
Understanding the intracellular signaling mechanism induced by ligand binding to the platelet vWF receptor, GPIb-IX, as well as platelet signaling in general, has been hampered by the lack of specific means to interfere with platelet signaling intracellularly at a molecular level. Studies on the GPIb-IXinduced platelet activation by biochemical approaches have shown that ligand binding to GPIb-IX induces a series of intracellular biochemical changes such as generation of thromboxane A2 (IIbß3 (
IIbß3 and GPIb-IX. In our CHO cell expression model, GPIb-IX mediates signaling leading to the activation of integrin
IIbß3 in a manner similar to that observed in platelets: (a) vWF binding to GPIb-IX in our CHO cell model not only induces integrin-vWF interaction but also induces soluble fibrinogen binding to the integrin
IIbß3, suggesting that GPIb-IX is unlikely to be simply presenting
IIbß3 to vWF or inducing changes in vWF, but is inducing a cellular signal that activates the ligand-binding function of the integrin (Fig 3). This is consistent with previous findings in platelets showing vWF binding to GPIb-IX initiates signaling leading to integrin
IIbß3 activation (
IIbß3 in CHO cells is thus significant to further understanding the GPIb-IXmediated signaling using specific molecular biological approaches.
In our CHO cell expression model, the vWF modulator, ristocetin, was used to induce binding of soluble vWF to GPIb-IX expressed in CHO cells. It is known that platelets do not bind to soluble vWF under physiological conditions. At the site of vascular injury, vWF binds to exposed subendothelial matrix proteins such as collagen. Collagen binding causes the exposure of the GPIb-IXbinding site in vWF probably by inducing a conformational change (IIbß3. Binding of vWF to platelets induced by ristocetin and other in vitro methods is similar to vWF binding induced by subendothelial matrix under flow conditions. In both cases vWF binds to essentially the same ligand-binding pocket on GPIb-IX in the NH2-terminal region of GPIb
, and can be inhibited by the same monoclonal antibodies (e.g., AK2) directed against the NH2-terminal region of GPIb-IX (
IIbß3 (
(
We have shown previously that 14-3-3, an intracellular signaling molecule, bound to the cytoplasmic domain of GPIb-IX, and that its binding was dependent upon the COOH-terminal region of GPIb (
inhibits GPIb-IXmediated
IIbß3 activation. As deletion of the COOH-terminal domain of GPIb
did not negatively affect vWF binding to GPIb-IX (Fig 6), it is unlikely that the inhibition in integrin activation resulted from a loss of vWF binding function of the mutant GPIb-IX. Since this GPIb-IX mutant still interacts with filamin at a nearby site (
cytoplasmic domain or loss of the interaction with the filamin-membrane skeleton caused the inhibition in signaling. Consistent with the importance of 14-3-3 in vWF-induced signaling, the small dominant negative fragment of 14-3-3 that contains the GPIb
-binding site (
Filamin binding to the central region of the GPIb cytoplasmic domain links GPIb-IX to the membrane skeleton structure (cross-linked short actin filaments) underlining the membrane (
at residue 559 abolished association of GPIb-IX with the filamin-membrane skeleton. Cells expressing this truncated mutant GPIb-IX spread on vWF without coexpression of
IIbß3 (
559/2b3a cells expressing this mutant GPIb-IX and integrin
IIbß3 showed an enhanced spreading on vWF which was poorly inhibited by anti-
IIbß3 antibodies that blocked ligand-binding sites but was significantly inhibited by RGDS peptide (Fig 7), suggesting that an RGDS-dependent endogenous integrin is responsible. In contrast to the truncation mutant, cells expressing wild-type GPIb-IX spread poorly on vWF in the absence of
IIbß3 (Fig 2). This suggests that the function of this endogenous CHO cell integrin to mediate cell spreading on vWF is restrained by the membrane skeleton association with GPIb-IX and enhanced by disruption of this association. When coexpressed with integrin
IIbß3, however, wild-type GPIb-IX is able to induce cell spreading on vWF (Fig 2) and soluble fibrinogen binding to integrin
IIbß3. Thus, GPIb-IXmediated activation of
IIbß3 is not restrained by the association of GPIb-IX with the membrane skeleton. This suggests that the functions of
IIbß3 and the endogenous integrin are regulated by GPIb-IX via different mechanisms. Indeed, we showed that GPIb-IXmediated activation of integrin
IIbß3 involves the binding of 14-3-3 to the COOH terminus of GPIb
. In contrast, the
559/2b3a cells expressing the mutant GPIb-IX lacking both filamin and 14-3-3binding sites were defective in vWF-induced fibrinogen binding (Fig 5). It remains unclear what types of endogenous CHO cell integrin are responsible for cell spreading on vWF in the absence of
IIbß3, and what mechanisms are involved in the upregulation of their function when GPIb-IX is dissociated from the membrane skeleton. CHO cells express an endogenous vitronectin receptor (
v complexed with ß1 or possibly ß5) and
5ß1, both of which are inhibited by RGDS peptides (
IIbß3 to interact with vWF and soluble fibrinogen is known to require prior activation via intracellular signaling (
IIbß3 is not restrained by the membrane skeleton structure, but requires a 14-3-3dependent signaling mechanism.
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Footnotes |
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1 Abbreviations used in this paper: GFP, green fluorescent protein; GP, glycoprotein; PGE1, prostaglandin E1; PKA, protein kinase A; vWF, von Willebrand factor.
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Acknowledgements |
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We thank Drs. Mark Ginsberg, Zaverio Ruggeri, Changeng Ruan, and Joan E.B. Fox for providing reagents and for discussions.
This work is in part supported by the grant HL52547 from National Institutes of Health, and by the National Heart Foundation of Australia. X. Du is an Established Investigator of the American Heart Association.
Submitted: 29 March 1999
Revised: 14 October 1999
Accepted: 18 October 1999
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References |
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