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Keywords:

  • von Willebrand factor;
  • fibrinogen;
  • adhesion;
  • αIIbβ3 integrin;
  • αvβ3 integrin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

Summary. Dithiothreitol (DTT) is known to induce an active conformation of αIIbβ3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-αIIbβ3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-αIIbβ3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-αIIbβ3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-αIIbβ3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50–1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated αIIbβ3 is absolutely required for spread cells to resist detachment and strengthens the importance of the αIIbβ3 activation step for adhesion and spreading to VWF.

Integrins are a family of cell surface αβ heterodimeric receptors that mediate cell extracellular matrix and cell–cell adhesion (Hynes, 1992). The β3 integrin family is composed of two members, αIIbβ3 and αvβ3. Both receptors share the same β3 subunit but have distinct α subunits, and interact with a variety of plasma proteins, as well as adhesive proteins of the extracellular matrix. In particular, β3 integrins are able to bind fibrinogen (Fg) and von Willebrand factor (VWF). Ligand binding to αIIbβ3 mediates platelet adhesion and aggregation, a key event in haemostasis and thrombosis, whereas binding to αvβ3 influences cell adhesion and regulates cell migration, particularly in atherosclerosis, restenosis, angiogenesis and tumour cell invasion.

αIIbβ3 is a receptor for Fg and VWF, which mediates platelet aggregation and spreading on the extracellular matrix. These responses are accompanied by major changes in platelet morphology and in the organization of the actin cytoskeleton leading to full spreading (Yuan et al, 1999). In resting platelets, unactivated αIIbβ3 integrin is able to support platelet adhesion to immobilized Fg (Kieffer et al, 1991). Signals generated through αIIbβ3 integrin after interaction with immobilized Fg cause major cytoskeletal rearrangement, leading to platelet spreading (Fox, 2001). Stimulation of platelets with various agonists such as thrombin or ADP will induce a conformational change in αIIbβ3 (referred to as inside-out signalling), which can then bind to soluble ligands such as Fg and VWF, as well as to immobilized VWF (Shattil et al, 1985; Parise, 1999). Moreover glycoprotein Ib (GPIb) binding to VWF at high shear rates may activate αIIbβ3, leading to platelet adhesion and aggregation on VWF (Savage et al, 1996). A requirement for αIIbβ3 has been further demonstrated for thrombus formation under flow conditions (Ruggeri et al, 1999; Tsuji et al, 1999). Activation of αIIbβ3 is a prerequisite for stable adhesion and signal transduction taking place on a VWF surface (Savage et al, 1992; Nesbitt et al, 2002). In contrast to VWF, activation of αIIbβ3 is not essential for platelet adhesion and spreading to Fg. Indeed, Fg contains two distinct binding sites that cooperate for optimal interaction, where one of them, called the dodecapeptide sequence, can bind to unactivated αIIbβ3 (Gartner et al, 1993).

Mild reduction in the presence of dithiothreitol (DTT) has been previously reported to promote Chinese hamster ovary (CHO)-αIIbβ3 cell aggregation in the presence of soluble Fg (Lyman et al, 1997). It has been shown that DTT reduces two disulphide bonds within the cysteine-rich domain of the β3 subunit, resulting in conformational changes leading to the opening of the RGD and dodecapeptide binding sites within both αIIb and β3 (Yan & Smith, 2001). However, these authors suggest that partial reduction by DTT leads to an intermediate activated state of αIIbβ3. We have recently established that in addition to Fg, DTT treatment enabled binding of CHO-αIIbβ3 cells to soluble as well as to immobilized VWF (Mekrache et al, 2002). Therefore, the question remained of the mechanism by which DTT-mediated αIIbβ3 activation could promote stable cell adhesion and spreading to immobilized VWF. Our results showed that αIIbβ3 activation by DTT was required to exhibit optimal CHO-αIIbβ3 cell spreading on VWF and that CHO-αIIbβ3 cells were able to adhere irreversibly to VWF and to resist to high shear rates under dynamic flow conditions.

Purification of adhesive proteins.  Human VWF and Fg were purified and depleted of contaminant fibronectin and, respectively, of Fg and VWF as described (Mekrache et al, 2002). Bovine serum albumin (BSA) was provided by Calbiochem (La Jolla, CA, USA) and was heat-denaturated at 60°C.

Monoclonal antibodies.  The αIIbβ3 complex-specific murine monoclonal antibody (mAb) AP2, was a generous gift from Dr T. J. Kunicki (The Scripps Research Institute, La Jolla, CA, USA) (Pidard et al, 1983). 10E5, which binds to αIIbβ3 (Coller et al, 1983), and 7E3 (Coller, 1985), which recognizes both αIIbβ3 and αvβ3, were provided by Dr B. Coller (SUNY Stony Brook, NY, USA). pl-55 is an anti-αIIbβ3-complex-specific mAb that only recognizes activated αIIbβ3 and was provided by Dr B. Steiner (Pharma Division, F. Hoffman-La Roche, Basel, Switzerland) (Steiner et al, 1993). All anti-αIIbβ3 antibodies used, except pl-55, bind to both active and inactive αIIbβ3. mAb 128, which recognizes the β3 subunit, and mAb9, directed against the VWF-RGD sequence, were produced in our laboratory by Dr J. P. Girma (Pietu et al, 1994; Veyradier et al, 1999). mAb LM609 (directed against the αvβ3 complex) was from Chemicon International (Temecula, CA, USA) (Cheresh & Spiro, 1987). Isotypic control antibodies were from Immunotech (Marseille, France). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab′)2 was purchased from Cappel (Turnhout, Belgium).

CHO-αIIbβ3 and CHO-αvβ3 cell culture.  For CHO-αIIbβ3 cells, the full-length cDNAs encoding for wild-type human αIIb or human β3 were inserted into the pBJ1 expression vector and co-transfected into CHO cells by the lipofectamine method, as described previously (Kieffer et al, 1996). Positive cells were selected for cell surface expression of the recombinant human integrin using the αIIbβ3 complex antibody AP2. Stably transfected cells were subcloned and analysed for cell surface expression of the transfected integrins with the αIIbβ3-complex-specific antibody PL2-73. Receptor density was 3000–5000 per cell, corresponding to a 10-fold lower density than in platelets. For CHO αvβ3 cells, the full-length cDNA encoding for wild-type human β3 was inserted into the pBJ1 expression vector and transfected into CHO cells by the lipofectamine method as described previously (Kieffer et al, 1996; Schaffner-Reckinger et al, 1998). Positive cells were analysed for cell surface expression of the recombinant human integrin β3 subunit using the anti-β3 mAb P37. Density of the chimaeric αvβ3 receptor was 3000–5000 per cell. Selected cell clones were grown in Iscove's medium (Eurobio, les Ulis, France) supplemented with 10% fetal calf serum (Boehringer Mannheim, Meylan, France), 2 mmol/l glutamine, 100 U/ml of penicillin and 100 µg/ml of streptomycin (Eurobio). Cells were grown to confluence and routinely passaged after detachment using EDTA (Carlo Erba, Val de Reuil, France) in 0·1 mmol/l Na2HPO4, 0·1 mmol/l KH2PO4, buffer pH 7·4 (phosphate-buffered saline, PBS).

Flow cytometry.  Flow cytometry was performed in a FACScan cytofluorimeter (Becton Dickinson, Le Pont-de-Claix, France). Briefly, CHO-αIIbβ3 cells or CHO-αvβ3 cells were harvested with 0·5 mmol/l EDTA from culture flasks and washed by centrifugation for 10 min at 25°C. They were resuspended at a concentration of 1·5 × 106/ml in HEPES buffer (10 mmol/l HEPES (N-[2-hydroxyethyl]piperazine-N′-[ethanesulphonic acid]), 140 mmol/l NaCl, 5·4 mmol/l KCl, 2 mmol/l CaCl2, 1 mmol/l MgCl2, 5·56 mmol/l glucose, pH 7·4) supplemented with 3% BSA. Then they were incubated with 20 µg/ml of primary mAb for 30 min at 4°C, washed with 0·1 mmol/l Na2HPO4, 0·1 mmol/l KH2PO4, buffer pH 7·4 (PBS) containing 0·1% BSA and incubated for 30 min at 4°C with a FITC-conjugated goat anti-mouse F(ab′)2. Cells were finally resuspended in 300 µl of PBS, 0·1% BSA. Fluorescence was detected by using the 520 nm band pass filter of the flow cytometer.

In some cases, CHO-αIIbβ3 cells were treated with 10 mmol/l DTT as described (Mekrache et al, 2002). CHO-αvβ3 were treated with 1 mmol/l MnCl2 for 5 min at room temperature, and then incubated with mAbs as described above.

Cell adhesion assay.  Confluent cells were washed, harvested with EDTA and centrifuged at 1100 r.p.m. for 10 min. CHO-αIIbβ3 cells were resuspended in HEPES buffer containing 3% BSA, and treated with buffer or 10 mmol/l DTT for 5 min at room temperature. After washing, they were used at 40 000 cells/ml. CHO αvβ3 cells were resuspended in HEPES buffer containing 3% BSA in the presence or in the absence of 1 mmol/l MnCl2. In some cases, cells were preincubated with 20 µg/ml of mAb for 30 min at 4°C. Cell adhesion was performed in 96-multiwell plastic plates (Dutscher, Brumath, France) coated overnight at 4°C with 10 µg/ml of VWF or 10 µg/ml of Fg, which were previously established as the concentrations allowing optimal adhesion of αvβ3 expressing cells in these wells (Perrault et al, 1997). Coated wells were rinsed before addition of 100 µl cell suspension incubated at 37°C for various times. Adherent cells were fixed and stained as described previously (Perrault et al, 1997). All assays were performed in duplicate and repeated at least three times. Adhesion to 20 µg/ml of BSA, used as a negative control, was always subtracted from the adhesion value to either VWF or Fg, and never exceeded 1–2% of the cell input in wells. Microphotographs of adherent cells were taken at a 20-fold magnification.

Quantification of cell adhesion and classification of adherent cells into attached or spread cells.  Quantification of adhesion was performed with a real-time digital imaging processing system (SAMBA 2005; Unilog Meylan, France) as described previously (Perrault et al, 1997). A minimum of nine fields was studied at a 10-fold magnification. The number of adherent cells per well was calculated from the average number of counted cells and normalized for the total surface of the well. Results are expressed as the percentage of adherent cells relative to the total number of deposited cells per well.

Classification of adherent cells into either attached or spread cells was based on morphological parameters recorded by image analysis as described previously (Perrault et al, 1998). Automatic classification was performed using a surface area threshold value of 350 µm2 for CHO αIIbβ3 cells, which distinguished attached cells from spread cells. Spread cells were defined as those with a higher surface area, while adherent cells with a lower surface area were classified as attached cells. No change of threshold was observed following cell activation by DTT. For CHO αvβ3 cells, the surface area threshold value was 250 µm2 as determined previously (Perrault et al, 1998). Results are expressed as the percentage of spread cells relative to the total number of deposited cells per well.

Means ± SEM were calculated for three experiments performed in duplicate. In some cases, statistical analysis was performed using Student's t-test for unpaired samples.

Cell adhesion assay: resistance to detachment by shear flow.  The flow chamber consisted of a rectangular cavity (0·2 mm deep, 29 mm long and 5 mm wide) carved in a plexiglas block (Billy et al, 1997). The bottom wall of the chamber was a glass coverslip of 0·17 mm3 × 60 mm3 × 24 mm3 (CML, Nemours, France) held in place by an aluminium plate, two metal clamping screws and an additional carved piece of aluminium on top. Controlled flow rate was generated with an electric pump (PHD 2000, Harvard Apparatus, les Ulis, France) using a 5- or 20-ml plastic syringe (Terumo, CML). All experiments were performed at room temperature. CHO αIIbβ3 cells, untreated or treated with 10 mmol/l DTT, were resuspended in Iscove's medium at a concentration of 800 000 cells/ml. They were allowed to adhere in the absence of flow for 30 min to a coverslip that had been precoated overnight with 40 µg/ml of purified VWF or Fg. Resistance to detachment by the adherent cells was assessed by perfusion of Iscove's medium through the chamber at different shear rates, which were increased every 5 min at 50, 100, 200, 400, 800 and 1600/s. The wall shear rate γ (/s) was calculated according to the function γ = 6Q/bd2, where Q is the volumetric flow rate (cm3/s), b (cm) the width and d (cm) the depth of the slit. The flow rate varied from 100 to 3200 µl/min, thus the wall shear rate varied from 50 to 1600/s. These levels of shear rates related to physiological levels found in vivo in the venous and arterial systems. The perfusion chamber was set on the stage of an inverted microscope (Axiovert 135, Zeiss, Germany) equipped with a 10× Hoffman Modulation Contrast objective. A charged-coupled-device (CCD) camera (Sony, Tokyo, Japan) and a 20-inch monitor (PVM20N2E, Sony) allowed real-time visualization of cell adhesion and detachment. A minimum of 12 fields were recorded with an S-VHS videotape recorder (SVO-9500MDP, Sony). Results were expressed as the percentage of remaining adherent cells as a function of adherent DTT-treated cells on Fg before any perfusion.

αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

Analysis of surface expression of αIIbβ3 in transfected CHO cell lines was performed by flow cytometry, using the anti-αIIbβ3 mAb AP2, the anti-β3 mAb 128, and the anti-αvβ3 mAb LM609 to detect the chimaeric (hamster αv)–(human β3) receptor (Table I). After DTT treatment, CHO-αIIbβ3 cells bound mAb pl-55, which only recognizes the activated αIIbβ3 complex (Mekrache et al, 2002). DTT treatment did not affect CHO-αIIbβ3 recognition by mAbs 128, AP2 and LM609. β3 integrin surface expression of CHO-αvβ3 cells was assessed using the anti-β3 mAb 128 and the anti-αvβ3 LM609 showing high levels of αvβ3 expression. No detectable αIIbβ3 receptor was observed in these cells. In the presence of Mn2+, no modification of αvβ3 expression was observed, indicating that the LM609 epitope was not influenced by activation (Table I).

Table I.  Flow cytometry analysis of Chinese hamster ovary (CHO) cells expressing αIIbβ3 or αvβ3 integrin.
Antibody used% of positive cells
CHO αIIbβ3CHO αIIbβ3 + DTTCHO αvβ3CHO αvβ3 + Mn2+
  1. Untreated and DTT-treated cells were incubated with 20 µg/ml of either control antibody, mAb 128 specific for the β3 subunit, AP2 specific for the αIIbβ3 complex, LM609 directed against the αvβ3 complex and pl-55 specific for the activated state of the αIIbβ3 receptor for 30 min at 4°C, washed and incubated with the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab′)2. Percentage of positive cells was expressed relative to the total cell number.

Control 7·6% 7·1% 8·6% 7%
mAb 12884%84%93·7%93·4%
AP292%87% 9·4% 8·6%
LM60955%42%90·1%91·9%
pl-5511%83% 8·9% 8·1%

Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

Mn2+ was previously described as an αvβ3 integrin activator (Stuiver et al, 1996), whereas DTT was shown previously to induce αIIbβ3 integrin activation (Kouns et al, 1994). To establish a model allowing the activation of either the αvβ3 or the αIIbβ3 complex, we evaluated the effect of activation of αvβ3 by Mn2+ or of αIIbβ3 cells by DTT on cell adhesion (Fig 1A–C) and spreading (Fig 1D–F).

image

Figure 1. Kinetics of Chinese hamster ovary (CHO)-αIIbβ3 and CHO-αvβ3 cell adhesion, and spreading on von Willebrand factor (VWF) or fibrinogen (Fg). Kinetics of adhesion (A–C), and spreading (D–F) were established for CHO-αIIbβ3 cells (B, C, E and F) or CHO-αvβ3 cells (A and D). Untreated CHO-αIIbβ3 (closed symbols) or cells treated by 10 mmol/l dithiothreitol (DTT) (open symbols) were allowed to adhere to VWF (B and E, symbols ▪ and □) or Fg (C and F, symbols ▴ and ▵) for different periods of up to 2 h; CHO-αvβ3 cells in the absence (▪) or in the presence of Mn2+ (□) were allowed to adhere to VWF for different periods of up to 120 min. Adherent cells were fixed and stained. Specific adhesion was calculated by subtracting non-specific adhesion measured on heat-denaturated bovine serum albumin (BSA)-coated wells. Results of cell adhesion, spreading are expressed as the percentage of adherent, spread cells relative to the total number deposited per well. Means ± SEM of three separate experiments performed in duplicate were reported.

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Adhesion of unactivated CHO-αvβ3 cell to VWF increased with time, reaching a value of 39·9 ± 7·3% at 60 min, which continued to increase up to 120 min. In contrast, in the presence of Mn2+, CHO-αvβ3 cell adhesion to VWF reached a plateau of 60·3 ± 5·6% at 60 min (Fig 1A). The effect of Mn2+ was even more striking on cell spreading: whereas the percentage of spreading of non-activated cells reached only 17·7 ± 2·6% at 60 min, after Mn2+ activation, a plateau of 45·7 ± 7·8% spread CHO-αvβ3 cells on VWF was had already been reached by 60 min (Fig 1D). Similar results were obtained when using Fg as a substrate (data not shown).

Differences in the kinetics of adhesion and spreading to VWF were also seen after DTT treatment of CHO-αIIbβ3 cells (Fig 1B and E). Adhesion of untreated cells to VWF increased in a time-dependent manner and tended to a plateau of 24·9 ± 3·2% at 60 min (Fig 1B). Interestingly, there was a twofold higher adhesion of DTT-treated cells compared with untreated cells (48·6 ± 9%) (Fig 1B). DTT treatment enhanced cell spreading to 32·1 ± 8·3% at 60 min, whereas there was hardly any detectable spreading of untreated cells (6·3 ± 1·3%) (Fig 1E). In contrast, DTT treatment was not required for αIIbβ3-dependent adhesion or spreading on Fg (Fig 1C and F) as a plateau of adhesion and spreading was reached at 40 and 60 min, respectively, independently of the addition of DTT. Only a slight increase (+10% of total) of adhesion and spreading was observed after DTT treatment. Altogether, these results showed that activation of αvβ3 by Mn2+ or of αIIbβ3 by DTT was associated with enhanced cell spreading on VWF. In contrast, adhesion and spreading of CHO-αIIbβ3 cells onto Fg was not significantly enhanced by DTT activation. We therefore selected cell spreading to VWF as a functional means to study αIIbβ3 integrin activation.

Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

To determine if the chimaeric αvβ3 receptor expressed on CHO-αIIbβ3 cells could contribute to adhesion and spreading of DTT-treated CHO-αIIbβ3 cells to VWF, mAbs directed against αIIbβ3 and/or αvβ3 were tested at 20 µg/ml for their ability to inhibit CHO-αIIbβ3 cell adhesion or spreading to VWF. We did not find any inhibitory effect of mAb LM609, a potent inhibitor of αvβ3 complex (Fig 2). In separate experiments, we verified that non-stimulated CHO-αvβ3 cell spreading on VWF was inhibited by LM609 (data not shown). CHO-αIIbβ3 cell adhesion and spreading on VWF was strongly inhibited by mAb 10E5 directed against the αIIbβ3 integrin, indicating the involvement of this receptor in DTT-treated CHO-αIIbβ3 cell adhesion to VWF. We also found that AP2 had an equivalent inhibitory effect as 10E5 on adhesion and spreading (data not shown). Interestingly, we found that mAb 7E3, which recognizes both αIIbβ3 and αvβ3 integrins, completely inhibited cell adhesion and spreading on VWF. This was confirmed by the combination of 10 µg/ml of each mAb 10E5 and LM609 (Fig 2). Altogether, these results suggest that following DTT treatment, CHO-αIIbβ3 cell adhesion and spreading on VWF predominantly involve αIIbβ3 and that blocking αIIbβ3 unmasks a secondary effect of αvβ3.

image

Figure 2. Effect of mAbs on CHO-αIIbβ3 cell adhesion and spreading on VWF. CHO-αIIbβ3 cells treated with 10 mmol/l DTT were preincubated for 30 min at 4°C with 20 µg/ml of either control mAb, 7E3 which recognizes both αIIbβ3 and αvβ3 integrins, 10E5 against the αIIbβ3 complex, LM609 directed against αvβ3 integrin, a mixture of 10E5 and LM609 (10 µg/ml each) or mAb 9 to VWF-RGD sequence. Cells were then allowed to adhere to VWF-coated wells for 1 h at 37°C, fixed and stained. Means ± SEM of adherent cells (black bars) or spread cells (grey bars) were calculated for three experiments performed in duplicate.

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To define the involvement of VWF-RGD sequence in DTT-treated cell adhesion to VWF, we used mAb 9, which was previously described as an inhibitor of VWF binding to platelet αIIbβ3 (Nokes et al, 1984). Results showed that this mAb was able to completely block CHO-αIIbβ3 cell adhesion and spreading on VWF (Fig 2). This was confirmed by showing that DTT-treated CHO-αIIbβ3 cells were unable to adhere and spread on rVWF-RGGS (data not shown), reported as unable to bind to thrombin-stimulated platelets (Lankhof et al, 1995).

Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

To evaluate the influence of DTT treatment on cell spreading on VWF, we assessed the effect of increasing DTT concentrations. We observed that spreading to Fg was independent of DTT treatment with 44·1 ± 4·9% spread cells in the absence of DTT and 42·7 ± 3·7% in the presence of 20 mmol/l DTT (Fig 3). Interestingly, DTT treatment induced an increase of CHO-αIIbβ3 cell spreading on VWF in a dose-dependent manner (Fig 3). Whereas CHO-αIIbβ3 cells were unable to spread on VWF in the absence of DTT (3·76 ± 2·8%), αIIbβ3 activation enhanced cell spreading up to 36·7 ± 2·8% for 20 mmol/l DTT. Thus, in contrast to Fg, VWF was suitable for the study of activated αIIbβ3-dependent adhesion and spreading.

image

Figure 3. Effect of DTT on CHO-αIIbβ3 cell spreading on VWF and Fg. Cells were preincubated with increasing concentrations of DTT for 5 min at room temperature. After washing, cells were resuspended in HEPES buffer and 4000 cells per well were allowed to adhere for 60 min at 37°C to VWF (▪) or Fg (▴). Quantification of cell spreading (▪, ▴) was measured by an automatic programme of cell counting and cell classification as described in Materials and methods. Results were expressed as the percentage of spread cells relative to the total number deposited per well. Means ± SEM of three separate experiments performed in duplicate were calculated.

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Interestingly, the morphology of cells adherent to Fg was quite different from that observed on VWF when compared at the same time (60 min). DTT-treated CHO-αIIbβ3 cells were able to adhere and a significant proportion of cells were beginning to spread on VWF (Fig 4A) whereas untreated cells were unable to spread and most of adherent cells which were observed remained in the typical round morphology of attached cells (Fig 4B). Adherent cells on Fg were completely spread independently of the presence of DTT (Fig 4C and D).

image

Figure 4. DTT-dependent CHO-αIIbβ3 adhesion to VWF and Fg. CHO-αIIbβ3 cells were detached and treated with 10 mmol/l DTT (A and C) or PBS (B and D) for 5 min. After washing, cells were resuspended in HEPES buffer and 4000 cells per well were allowed to adhere to VWF (10 µg/ml) (A and B) or Fg (C and D) for 60 min at 37°C. Adherent cells were fixed and stained. Microphotographs of cell adhesion were taken at 20-fold magnification.

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To better define these differences, we calculated the surface area of spread cells using automated image analysis (Fig 5). Interestingly, we observed that the surface area of spread cells on Fg was significantly higher than that observed on VWF (P < 0·05). Furthermore, this difference increased in a time-dependent manner (Fig 5). Our results showed that activation of αIIbβ3 was required to induce optimal cell spreading to VWF, whereas cell adhesion and spreading to Fg was already complete and independent of receptor activation.

image

Figure 5. Surface area comparison of spread CHO-αIIbβ3 cells treated with DTT on VWF and Fg. After cell adhesion to VWF, the morphology of adherent cells on VWF (▪) or Fg (▴) was evaluated by image analysis. At each time-point, statistical comparison of surface area was performed between spread cells on VWF and Fg using Student's t-test for unpaired samples. Data are means ± SEM and were calculated for three experiments performed in duplicate.

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Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

We next wanted to evaluate whether these morphological differences between VWF and Fg could reflect the strength of the interaction between activated αIIbβ3 receptor and its ligands, VWF and Fg. To this end, we assessed the ability of adherent and spread cells to resist increasing shear rates during perfusion with a cell-free medium. Interestingly, DTT treatment allowed more stable adhesion on VWF as compared with untreated cells, as shown by 62·4 ± 8·6% of cells remaining adherent following increasing shear rates from 50 to 1600/s. In contrast, a lower proportion (15·1 ± 9·4%) of untreated cells remained adherent on VWF, suggesting that low-affinity interactions were already abolished in the presence of low shear rates (Fig 6). No CHO-αIIbβ3 cell displacement was observed on Fg, showing that CHO-αIIbβ3 adhesion to Fg could resist increasing shear rates, independently of DTT treatment. These results were in agreement with the morphological differences reported in static conditions (Fig 4) as spread cells continued to exhibit a higher extent of spreading on Fg than on VWF after perfusion with cell-free medium at high shear rates.

image

Figure 6. Effect of increasing shear rate on CHO-αIIbβ3 cell detachment from VWF or Fg. DTT-treated (□, ▵) or untreated CHO-αIIbβ3 cells (▪, ▴) were allowed to adhere to 40 µg/ml of VWF (□, ▪) or Fg (▵, ▴) for 30 min at room temperature without any perfusion. Cells were then submitted to increased shear rates with Iscove's medium up to a maximum of 1600/s. Adherent cells were counted in each field and the average of adherent cells in each shear condition was evaluated and expressed as the percentage of residual adherent cells as a function DTT-treated cell adhesion to Fg in the absence of perfusion. Results are means ± SEM of three experiments.

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Our results suggest that once treated with DTT, CHO-αIIbβ3 adherent cells to VWF were able to resist high shear rates to a similar extent as adhesion to Fg. In contrast, untreated cells were unable to remain adherent to VWF, whereas 100% of these cells remained adherent to Fg in the presence of increasing shear rates.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

In the present study we have demonstrated that DTT-activation of CHO-αIIbβ3 cell was required to induce significant CHO-αIIbβ3 cell adhesion and spreading on VWF, in static conditions. In addition, we have found that DTT activation promoted stable adhesion on immobilized VWF allowing adherent cells to resist detachment generated by high shear rates.

In the absence of DTT treatment, CHO-αIIbβ3 cells expressed the receptor in a low affinity state, which allowed adhesion and spreading to Fg, but was unable to induce cell spreading on VWF. Our present results show that DTT treatment enhanced firm CHO-αIIbβ3 cell adhesion and spreading onto VWF in a dose-dependent manner, presumably in relation to the exposure of an epitope recognized by mAb pl-55, specific for activated αIIbβ3. The fact that spreading of DTT-treated CHO-αIIbβ3 on VWF predominantly involves αIIbβ3 was further demonstrated by the inhibitory effect of mAb 10E5 to αIIbβ3 as well as mAb 9 to the VWF-RGD sequence. Although chimaeric αvβ3 integrin was expressed at the surface of CHO-αIIbβ3 cells, we have found that it did not play a role in DTT-treated CHO-αIIbβ3 cell adhesion to VWF, unless the αIIbβ3 receptor was blocked. In contrast, we have confirmed that Mn2+ modulates αvβ3 integrin activity and promotes CHO-αvβ3 cell spreading on VWF, a finding already established for adhesion to vitronectin (Stuiver et al, 1996). Thus, in the absence of Mn2+, the contribution of αvβ3 integrin in DTT-treated CHO-αIIbβ3 cell adhesion to VWF remained minimal.

This effect of DTT on cell spreading onto VWF represents thus a new functional assay for activated state of αIIbβ3. DTT has been previously reported to promote CHO-αIIbβ3 cell aggregation in the presence of soluble Fg (Lyman et al, 1997) or soluble VWF (Mekrache et al, 2002). Other tools used to activate the αIIbβ3 integrin in cellular models are essentially anti-LIBS mAbs, which have been shown to promote cell aggregation in the presence of Fg (Frelinger et al, 1991; Lyman et al, 1997) or cell adhesion to VWF (Hato et al, 1998).

The mechanism by which DTT could induce an activated conformation through partial reduction of disulphide bonds in αIIb and/or β3, has been recently elucidated. By comparing the resting and active forms of αIIbβ3 by high resolution peptide mapping, Yan et al (2000) identified differences about disulphide bonds in the extracellular domain of β3. These two forms have a different number of free cysteine residues indicating that an overall net reduction is correlated with a change in activation status (Yan & Smith, 2001). Integrin activation could be controlled directly by a redox site in the extracellular domain, independently of inside-out signalling (Yan & Smith, 2000). This idea is supported by the finding that platelets have a protein-disulphide isomerase activity, which may be involved in αIIbβ3 activation (Essex & Li, 1999; O'Neill et al, 2000). Furthermore, there is evidence of natural mutations of β3 within the cysteine-rich domain causing activation of αIIbβ3 (Wippler et al, 1994; Kashiwagi et al, 1999). Such a mutation may be found associated with a thrombasthenic phenotype (Ruiz et al, 2001). In particular, the C598Y mutation induced spontaneous PAC-1 binding of CHO-αIIbβ3 cells, suggesting that Cys598 is potentially involved in disulphide bond reshuffling (Chen et al, 2001).

Whereas Fg has been widely used to promote cell–cell aggregation, it does not distinguish between unstimulated and stimulated CHO-αIIbβ3 cell adhesion. This is largely due to its properties as a substrate for cell adhesion. Indeed, αIIbβ3 integrin activation was not required to promote platelet or CHO-αIIbβ3 cell adhesion to Fg (Gartner et al, 1993; Farrell & Thiagarajan, 1994). We found that DTT treatment did not affect CHO-αIIbβ3 cell adhesion and spreading to Fg. In contrast, immobilized VWF did not support any CHO-αIIbβ3 cell adhesion and spreading unless the receptor was activated by DTT.

We have used a small perfusion chamber coupled to videomicroscopy to confirm this finding, namely that DTT was absolutely required to activate αIIbβ3, thus enabling adherent and spread CHO-αIIbβ3 cells to remain on the VWF surface when submitted to rinsing with a cell-free medium under increasing flow rates. In contrast, no difference was observed for cells spread on Fg whether or not DTT was used to activate the receptor, as untreated or DTT-treated cells were able to resist to extremely high flow rates. Interestingly, in both static and flow conditions, we found quantitative differences in cell surface area, which was significantly lower on VWF than on Fg. This may be related to the slight decrease in the percentage of DTT-treated cells remaining adherent to VWF under flow, whereas the percentage was kept constant on Fg.

Resistance to detachment by high shear rates has been previously shown in platelets or CHO cells expressing GPIb in combination with αIIbβ3 (Savage et al, 1992; Yap et al, 2000). However, in both models GPIb was present and required to support initial contact and tethering. It was shown that blocking of αIIbβ3 resulted in a loss of stable adhesion. Thus, we present the first evidence that in flow conditions, VWF-adherent CHO-αIIbβ3 cells that expressed activated αIIbβ3, were able to resist high shear rates independently of the presence of GPIb, confirming the critical role of αIIbβ3 in mediating stationary cell adhesion.

The present cellular model of αIIbβ3 interaction with immobilized VWF in the absence of GPIb, has demonstrated that irreversible adhesion, which was resistant to flow conditions, did not occur in the absence of αIIbβ3 activation. It is interesting to note that αIIbβ3/VWF interaction is necessary for stable adhesion and thrombus formation at high shear rates (Ruggeri et al, 1999; Yap et al, 2000; Savage et al, 2001). Under high shear rates, GPIb must be present to allow the slowing down of platelets at the VWF surface, thus establishing the first contact between platelet and VWF. Numerous studies have focused on the process during which platelet αIIbβ3 becomes activated (Savage et al, 1996; Tsuji et al, 1996; Kulkarni et al, 2000). It has become clear that signalling mechanisms result from GPIb/VWF interaction and lead to αIIbβ3 activation such as calcium signalling, a process at least partially associated with PI-3 kinase activation, which is a strong inducer of platelet spreading (Nesbitt et al, 2002; Yap et al, 2002). Our data, showing that activated αIIbβ3 is absolutely required for spread cells to resist detachment, strengthens the importance of this αIIbβ3 activation step, in a manner which is relatively independent of the extent of shear rates.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References

We thank Dr T. Kunicki, Dr B. Coller, Dr M. Ginsberg, Dr B. Steiner for the gift of monoclonal antibodies, Dr C. Perrault for help in image analysis and Dr T. Lindhout for providing the flow chamber. This study was supported by EC Biomed No. CT98-3517 (D.B. and N.K.). M.M. was supported by Sanofi Association for Thrombosis Research and the French Society of Atherosclerosis.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. αIIbβ3 and αvβ3 Integrin expression at the surface of CHO cells
  6. Activation αIIbβ3 or αvβ3 integrin regulates CHO cell spreading
  7. Inhibition of DTT-stimulated CHO-αIIbβ3 cell adhesion and spreading on VWF
  8. Requirement of DTT for CHO-αIIbβ3 cell spreading on VWF but not on Fg
  9. Requirement of DTT for VWF-adherent CHO-αIIbβ3 cell resistance to detachment under flow conditions
  10. Discussion
  11. Acknowledgments
  12. References
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