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Prepublished online as a Blood First Edition Paper on May 24, 2002; DOI 10.1182/blood-2002-02-0418.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Blood Research Institute, Blood Center of
Southeastern Wisconsin, and the Cardiovascular Center, Departments of
Pharmacology and Cellular Biology, Medical College of Wisconsin,
Milwaukee.
The major platelet integrin Integrins, one of several gene families that encode
cell surface adhesive receptors capable of mediating cell-cell and
cell-extracellular matrix (ECM) interactions,1 are
heterodimers consisting of a 120- to 180-kDa The platelet glycoprotein (GP) GPIIb-IIIa complex (integrin
GPIIIa (the integrin A number of gain-of-function GPIIIa mutations have been experimentally
induced and studied in recombinant GPIIb-IIIa-transfected cells. Bajt
et al demonstrated that replacement of residues 129 to 133 within the
ligand-binding site of GPIIIa with the corresponding sequence from the
integrin Materials
Cells and cell lines
Western blot analysis of expressed GPIIIa Stable CHO cell lines expressing WT GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa were harvested, washed, and solubilized in lysis buffer either containing 50 mM Tris (tris(hydroxymethyl)aminomethane), 2% sodium dodecyl sulfate (SDS), 10 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 100 µg/mL leupeptin (for nonreducing condition), or containing 50 mM Tris, 2% SDS, and 5% -mercaptoethanol (for reducing condition) at 4°C.
After centrifugation at 15 000g for 30 minutes, soluble
lysates were electrophoresed on 7% SDS-polyacrylamide gel
electrophoresis (PAGE), and transferred to polyvinylidene difluoride
(PVDF) membranes (Millipore). The membranes were blocked with
3% bovine serum albumin (BSA) and incubated with well-characterized
rabbit polyclonal antibodies specific for GPIIIa.34 After
washing, the membrane was incubated with goat-anti-rabbit IgG
conjugated with horseradish peroxidase, followed by chemiluminescence
detection according to the manufacturer's instructions (Amersham Life
Science, Piscataway, NJ).
Immunoprecipitation of GPIIb-IIIa complex from lysates of the transfected CHO cells Nontransfected CHO cells and transfected CHO cells expressing WT GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa were surface-labeled with 5 mM NHS-LC-biotin (Pierce, Rockford, IL) in phosphate-buffered saline (PBS) for 30 minutes at 22°C, and solubilized in lysis buffer (20 mM Tris, 100 mM NaCl, 1% Triton X-100, 2 mM PMSF, and 100 µg/mL leupeptin) for 30 minutes on ice.35 The supernatant was obtained by centrifugation at 15 000g for 30 minutes at 4°C. Aliquots of biotin-labeled cell lysates were precleared, and incubated overnight at 4°C with monoclonal antibodies (mAbs) specific for GPIIIa subunit, the GPIIb-IIIa complex, or normal mouse IgG (NMIgG). Rabbit anti-mouse IgG was added, and the immune complexes were recovered with protein A Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). The beads were washed with lysis buffer and resuspended in reducing sample buffer. The boiled samples were separated on 7% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 3% BSA in triethanolamine-buffered saline (TBS) overnight at 4°C. After washing, the membrane was incubated for 60 minutes with streptavidin conjugated with horseradish peroxidase, washed, and detected by enhanced chemiluminescence (ECL) as described above.Flow cytometric analysis Nontransfected and transfected CHO cells were harvested, washed, and incubated on ice with mAbs specific for each subunit, the GPIIb-IIIa complex, or NMIgG at a final concentration of 40 µg/mL. After 60 minutes of incubation, the samples were washed and incubated with a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse IgG (Jackson Lab, West Grove, PA) for 60 minutes. NMIgM and FITC-conjugated goat-anti-mouse IgM (Jackson Lab) were used as the negative control and the secondary antibody for PAC-1 (mIgM). The samples were washed and subjected to flow cytometric analysis using a FACScan (Becton Dickinson). Selected flow cytometric analysis was performed in the presence of RGDW peptide (1 mM), using RGEW at the same concentration as a negative control.Quantitation of soluble fibrinogen binding to GPIIb-IIIa on transfected CHO cells Fibrinogen was labeled with FITC using a protocol provided by Dr Paul F. Bray (Johns Hopkins University, Baltimore, MD). Briefly, 4 mg fibrinogen in 0.1 M sodium bicarbonate buffer, pH 9.0, was mixed with 20 µg 10% FITC dispersed on diatomaceous earth (FITC-Celite, Molecular Probes, Eugene, OR) and incubated for 30 to 60 minutes at 22°C in the dark. The labeling reaction was stopped by adding 0.1 mL freshly prepared 1 M ammonium bicarbonate, pH 8.0, for 10 minutes. Labeled fibrinogen was separated from Celite by centrifugation, and excess FITC was removed by dialysis against PBS overnight at 4°C in the dark. Fibrinogen concentration was determined with the BCA protein assay (Pierce). The ratio of FITC to fibrinogen was determined by comparing A280 to A493. After sterile filtration, FITC-conjugated fibrinogen was stored at 4°C until use. FITC-labeled BSA was prepared for use as a control for background binding. Nontransfected and transfected CHO cells were washed and incubated at 22°C with FITC-conjugated fibrinogen at a final concentration of 100 µg/mL in Hanks balanced salt solution (HBSS) containing Ca++ and Mg++ (1 mM). After incubation for 60 minutes, the samples were then washed, diluted, and subjected to flow cytometric analysis.Cell adhesion assays Cell adhesion assays were performed using vital dye-labeled cells as described previously.36 The 96-well plates (Immunlon 2, Dynatech Labs, Chantilly, VA) were coated overnight at 4°C with 0.1 mL PBS containing different concentrations of fibrinogen (2.5-10 µg/mL), 10 µg/mL AP2, or 1% BSA. The wells were then washed twice with PBS and blocked with 1% BSA in PBS at 22°C for 60 minutes. Transfected CHO cells were harvested, washed twice with MEM, and labeled with 2 µM calcein AM (Molecular Probes) at 37°C for 30 minutes. After washing with HBSS, labeled cells were counted and suspended in -MEM media (serum-free) at a concentration
of 1 to 2 × 106/mL, then added to each well
(1-2 × 105/well) and incubated for 60 minutes at 37°C.
Nonadherent cells were removed by washing twice with -MEM media;
adherent cells in each well were examined by microscopy and quantified
using a microplate fluorescence reader (CytoFluor II, Perseptive
Biosystem, Bedford, MA) at an excitation wavelength 485 nm and an
emission wavelength 530 nm. The fluorescence intensity of each well was measured before washing as the total cells added and that after washing
as the adherent cells. Quantitative data of cell adhesion were
expressed as the percentage of the total cells added with that remained
in each well after washing. All experiments were performed in
triplicate and repeated at least 3 times. In selected cell adhesion
assays, cells were preincubated with mAbs (40 µg/mL) or with RGDW and
RGEW peptides (2 mM) at 22°C for 20 minutes.
Tyrosine phosphorylation of pp125FAK Suspended cells were seeded onto plastic dishes that had been precoated with 10 µg/mL fibrinogen. After incubation for 30, 60, or 90 minutes at 37°C, plates were washed twice with ice-cold PBS. Then adherent cells were lysed on the plates with Triton lysis buffer containing sodium vanadate (1 mM) and scraped into microcentrifuge tubes. Lysates were incubated on ice for 30 minutes and clarified supernatants were processed for pp125FAK immunoprecipitation using a rabbit polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), and protein-A Sepharose (Pharmacia). Precipitates were separated on 7% SDS-PAGE and transferred to a PVDF membrane. Phosphotyrosine was detected with mAb, PY20.
The long-range disulfide bond between residues Cys5 and Cys435 of GPIIIa is disrupted by Cys5Ala or by Cys435Ala substitution The CHO cells transfected with GPIIb and either WT, Ala5GPIIIa, or Ala435GPIIIa were established. The presence of these mutations was confirmed by DNA sequence analysis (Figure 1A). Cys5 and Cys435 have been reported to form a disulfide bridge that brings the N-terminal region and EGF repeats of GPIIIa into close physical proximity.15 Substitution of either cysteine 5 or 435 with alanine would be predicted to result in conformational change, which should be visible as a shift in the migration of GPIIIa on a nonreduced SDS-PAGE gel. As shown in Figure 1B, the WT, Ala5, and Ala435 forms of GPIIIa run with the same mobility under reducing conditions. However, under nonreducing conditions, both Ala5GPIIIa and Ala435GPIIIa migrate more slowly than does WT GPIIIa. These data indicate that the conformation of GPIIIa has been altered by the alanine 5 or 435 mutations.
Ala5GPIIIa and Ala435GPIIIa are capable of associating with GPIIb and the integrins are expressed normally on the cell surface The ability of Ala435GPIIIa to associate with GPIIb is shown in Figure 2A. Both AP3 (specific for the GPIIIa subunit) and AP2 (specific for the GPIIb-IIIa complex) immunoprecipitated GPIIb-Ala435GPIIIa together, demonstrating that the formation of an integrin complex takes place. Essentially the same results were seen for the Ala5GPIIIa variant (not shown). The expression of GPIIb-Ala5GPIIIa and GPIIb-Ala435IIIa complexes on the cell surface was analyzed by flow cytometry using AP2 and AP3 (Figure 2B), and as shown, these 2 antibodies specifically and positively stained CHO cells with similar mean fluorescence intensities. Together, these data demonstrate that neither Cys5Ala nor Cys435Ala mutations affect the ability of GPIIIa to associate with GPIIb within cells, and that both Ala5GPIIIa and Ala435GPIIIa are expressed with GPIIb at normal levels on the cell surface. These cloned CHO cell lines, which express nearly equivalent amounts of WT GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa were used in the subsequent analyses.
The conformation of the GPIIb-IIIa complex is altered by the Cys5Ala and Cys435Ala mutations To examine whether the Cys5Ala or Cys435Ala substitutions affect the conformation of GPIIb-IIIa on the CHO cell surface, we performed flow cytometric analysis on transfected CHO cells using AP5 and D3,9,27 each of which binds to LIBS determinants on the activated form of GPIIb-IIIa complex. The binding of AP5 and D3 to CHO cells expressing WT GPIIb-IIIa was low (Figure 3A, left) as expected. In contrast, the binding of LIBS antibody AP5 to CHO cells expressing GPIIb-Ala435IIIa (Figure 3A, upper right), and the binding of LIBS antibody D3 to CHO cells expressing GPIIb-Ala5IIIa were constitutively high (Figure 3A, lower middle). AP5 could not report the conformational change elicited by the Cys5Ala mutation, as its epitope (GPIIIa residues 1-6)9 is proximal to the amino acid substitution. Similarly, the ability of D3 to bind the 435AlaGPIIIa variant is lost due to the fact that its epitope is near residue 422 of GPIIIa.37 However, the binding of non-LIBS antibody, AP3, which mapped within the residues 348-421 of GPIIIa, was not affected by Cys435Ala substitution (Figure 2B). The activation index of other LIBS antibodies is summarized in Figure 3B. Taken together, these data suggest that the conformation of both the Cys5Ala and Cys435Ala forms of the GPIIb-IIIa is altered.
Both GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes constitutively exist in a ligand binding-competent state To assess the ability of the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes to bind ligand, we examined the binding of the ligand-mimetic antibodies Pl-5531 and PAC-129,30 to transfected CHO cell lines. Although Pl-55 bound poorly to CHO cells expressing WT GPIIb-IIIa (Figure 4A), it bound avidly to CHO cells expressing GPIIb-Ala5IIIa or GPIIb-Ala435IIIa complexes. Similar results were obtained using PAC-1 (Figure 4B, upper). Binding was specific because it could be completely inhibited by excess RGDW peptide (Figure 4B, lower). These data demonstrate that the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes exist in an activated, ligand binding-competent state.
Both GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes exhibit constitutive fibrinogen binding To provide further evidence that the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes exist in an activated state, their ability to bind soluble fibrinogen was assessed. As shown in Figure 5, FITC-conjugated fibrinogen bound to GPIIb-Ala435IIIa-transfected CHO cells nearly 2-fold better than it did to CHO cells expressing WT GPIIb-IIIa. The binding of FITC-conjugated fibrinogen to GPIIb-Ala435IIIa-transfected CHO cells could be blocked by RGDW peptide (2 mM), but RGEW peptide at the same concentration did not inhibit this binding. The adhesive properties of the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes were further examined using immobilized fibrinogen. CHO cells expressing GPIIb-Ala5IIIa or GPIIb-Ala435IIIa complexes bound and spread on microtiter wells that had been coated with either low (2.5 µg/mL) or high (10 µg/mL) concentration of immobilized fibrinogen (Figure 6A), whereas CHO cells expressing WT GPIIb-IIIa failed to adhere to immobilized fibrinogen at low concentration (Figure 6A, top). The quantitative results of the cell adhesion are shown in Figure 6B. In addition, whereas LIBS antibodies D3, AP5, and 7G2, significantly increased the adhesion of WT GPIIb-IIIa transfectants to low-density immobilized fibrinogen (Figure 6C), they had little additional effect on the already increased adhesion of CHO cells expressing GPIIb-Ala5IIIa or GPIIb-Ala435IIIa. Taken together, these data provide evidence that the Ala5 and Ala435 mutations result in a conformationally altered high-affinity GPIIb-IIIa complex.
Tyrosine phosphorylation of pp125FAK in transfected CHO cells Ligand binding and clustering of integrins stimulate outside-in signaling, manifested by responses that include protein tyrosine phosphorylation and cytoskeletal reorganization. FAK, a 125-kDa cytoplasmic tyrosine kinase, is a component of focal adhesions and is a well-established component of integrin signaling pathways.8 To assess whether the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes are capable of mediating outside-in signaling response on cell adhesion, the tyrosine phosphorylation state of pp125FAK in CHO cells expressing WT GPIIb-IIIa, GPIIb-Ala5IIIa, and GPIIb-Ala435IIIa was compared. As shown in Figure 7, pp125FAK was not tyrosine-phosphorylated in nontransfected CHO cells after incubation on immobilized fibrinogen. In contrast, GPIIb-Ala5IIIa and GPIIb-Ala435IIIa, as well as WT GPIIb-IIIa, transfectants exhibited pp125FAK phosphorylation when bound to immobilized fibrinogen, indicating that these active mutant receptors are able to mediate outside-in signaling response on cell adhesion.
In this report, we examine the effect of Cys5Ala and Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that (1) both Ala5GPIIIa and Ala435GPIIIa are capable of associating with GPIIb and are expressed normally on the surface of transfected CHO cells; (2) both GPIIb-Ala5GPIIIa and GPIIb-Ala435IIIa exist in an activated conformational state, as reported by the constitutive binding of LIBS antibodies such as AP5 or D3, ligand-mimetic antibodies such as PAC-1 and Pl-55, and soluble fibrinogen; and (3) as a consequence of its activated state, both GPIIb-Ala5GPIIIa and GPIIb-Ala435IIIa confer to transfected CHO cells high-affinity ligand-binding properties on immobilized fibrinogen. Because both Ala5 and Ala435 mutations in GPIIIa result in similar effects on the adhesive properties of GPIIb-IIIa, our studies provide functional confirmation for the presence of a long-range disulfide bond between Cys5-Cys435,15 and support the notion that this region participates in the conformational changes associated with receptor activation. Additionally, these data provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.38,39 GPIIIa contains 56 cysteines, 7 of which are located within the
still-to-be-visualized PSI domain, 4 within the In platelet functional assays, it has been previously shown that
treatment of platelets with the reducing agent dithiothreitol induces
platelet aggregation, fibrinogen binding, and GPIIb-IIIa conformational
changes.38,39 Similar changes have been observed in other
integrins.40 The studies reported here demonstrate that
genetic disruption of Cys5-Cys435 is sufficient
to activate the complex. Interestingly, disruption of other disulfide
bonds near or within the cysteine-rich EGF repeat region of the
molecule, including those at Cys In addition to their physiologic role, both GPIIb and GPIIIa are known
to bear a number of clinically important alloantigenic determinants,
such as PlA1, the expression of which is controlled by a
Leu33Pro substitution within the PSI domain at the N-terminus of
GPIIIa. We have previously shown that disruption of the long-range
Cys5-Cys435 disulfide bond results in the
production of GPIIIa isoforms that bind some, but not all,
anti-PlA1 alloantibodies, suggesting that mutations in this
long-range disulfide bond can alter the conformation of GPIIIa. This
substitution also resulted in the loss of the epitope for certain LIBS
mAbs as reported here and previously,43 consistent with
the data that some LIBS antibodies recognize epitopes within the EGF
domains of GPIIIa.6 Because the
Cys5-Cys435 disulfide bridge of GPIIIa connects
the PSI domain to a region immediately amino terminal to the
cysteine-rich EGF repeat region of GPIIIa, and brings the N-terminal
region and the EGF domains of GPIIIa into physical proximity, this
disulfide bridge may have the potential to regulate the shape of the
ligand-binding pocket, and thereby affect the affinity of GPIIb-IIIa
for its ligands. In the crystal structure, the GPIIb-IIIa has been reported to contain endogenous thiol isomerase
activity, predicted from the presence of the tetrapeptide motif, CXXC,
in each of the EGF domains of One fundamental function of integrins is ligand binding, which in many
cases is regulated by a process referred to as inside-out signaling or
integrin activation.4,5 The importance of rapid regulated
changes in integrin affinity/avidity is easy to appreciate for
GPIIb-IIIa because platelets must interact productively with fibrinogen
or VWF following in vascular injury. The significance of inside-out
signaling and, in particular, affinity modulation for
We are grateful to Drs Eric Brown, Lisa Jennings, Alexy Mazurov, Sandy Shattil, Beat Steiner, and Nathalie Valentin for the generous supply of monoclonal antibodies used in this study.
Submitted February 28, 2002; accepted May 6, 2002.
Prepublished online as Blood First Edition Paper, May 24, 2002; DOI 10.1182/blood-2002-02-0418.
Supported by Grant-in-Aid Award 0050581N (Q-H.S.) from the American Heart Association, and by Program Project grant P01 HL44612-12 (P.J.N.) from the National Institutes of Health.
Q-H.S. and C-Y.L. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Qi-Hong Sun, Blood Research Institute, The Blood Center of Southeastern Wisconsin, 8727 Watertown Plank Rd, PO Box 2178, Milwaukee, WI 53233; e-mail: qsun{at}bcsew.edu.
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 A. L. Jannuzi, T. A. Bunch, R. F. West, and D. L. Brower Identification of Integrin {beta} Subunit Mutations that Alter Heterodimer Function In Situ Mol. Biol. Cell, August 1, 2004; 15(8): 3829 - 3840. [Abstract] [Full Text] [PDF]
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A. Chigaev, G. J. Zwartz, T. Buranda, B. S. Edwards, E. R. Prossnitz, and L. A. Sklar Conformational Regulation of {alpha}4{beta}1-Integrin Affinity by Reducing Agents: "INSIDE-OUT" SIGNALING IS INDEPENDENT OF AND ADDITIVE TO REDUCTION-REGULATED INTEGRIN ACTIVATION J. Biol. Chem., July 30, 2004; 279(31): 32435 - 32443. [Abstract] [Full Text] [PDF]
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 N. Butta, E. G. Arias-Salgado, C. Gonzalez-Manchon, M. Ferrer, S. Larrucea, M. S. Ayuso, and R. Parrilla Disruption of the {beta}3 663-687 disulfide bridge confers constitutive activity to {beta}3 integrins Blood, October 1, 2003; 102(7): 2491 - 2497. [Abstract] [Full Text] [PDF]
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 M. J. Quinn, T. V. Byzova, J. Qin, E. J. Topol, and E. F. Plow Integrin {alpha}IIb{beta}3 and Its Antagonism Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 945 - 952. [Abstract] [Full Text] [PDF]
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C. Buensuceso, M. de Virgilio, and S. J. Shattil Detection of Integrin alpha IIbbeta 3 Clustering in Living Cells J. Biol. Chem., April 18, 2003; 278(17): 15217 - 15224. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
IIb
3) integrin complexes
Top
Abstract
Introduction
7 within the
disordered PSI domain, and 3 within linker 2 (residues 435-452). Cys5 and Cys435 are found within the PSI and
linker 2 regions, respectively, and if paired as
proposed,
Ala mutations
in either of these residues have similar effects on complex formation,
surface expression, and cell adhesion, our studies provide functional
evidence that a long-range disulfide bond between
Cys5-Cys435 may indeed exist.
