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Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

Gu M, Xi X, Englund GD, Berndt MC, Du X - J. Cell Biol. (1999)

Bottom Line: 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 GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3).Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ilinois 60612, USA.

ABSTRACT
We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) 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 GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

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Adhesion of cells expressing recombinant receptors for vWF. (A) Cells expressing integrin αIIbβ3 (2b3a), cells expressing GPIb-IX (1b9) and cells expressing both GPIb-IX and αIIbβ3 (123) are described in Materials and Methods. These cells were detached and incubated with biotin-labeled monoclonal antibody WM23 (against GPIbα) at 22°C for 30 min, and then incubated with phycoerythrin-labeled streptavidin and FITC-labeled monoclonal antibody D57 (against integrin αIIbβ3) at 22°C for 30 min. The cells were then analyzed by flow cytometry. (B) The cells described in A were incubated in vWF-coated microtiter wells for 30 min at 37°C in the presence of 5 μg/ml botrocetin. Untransfected CHO cells were also used as a negative control. To compare the function of αIIbβ3 expressed in 123 and 2b3a cells, cells are also incubated with fibrinogen (Fg)-coated wells. After washing, the adherent cells were quantitated by an acid phosphatase assay as described in Materials and Methods. Shown in the figure are the results from triplicate samples (mean ± SD). (C) 123 cells, 2b3a and CHO cells were also allowed to adhere to (10 μg/ml) vWF-coated microtiter wells in the absence of botrocetin. 123 cells were also preincubated with control IgG, or the monoclonal antibodies, AK2 and SZ2 against the vWF-binding site of GPIbα, and then allowed to adhere to vWF-coated wells in the absence of botrocetin. Note that AK2 and SZ2 inhibited cell adhesion to vWF.
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Figure 1: Adhesion of cells expressing recombinant receptors for vWF. (A) Cells expressing integrin αIIbβ3 (2b3a), cells expressing GPIb-IX (1b9) and cells expressing both GPIb-IX and αIIbβ3 (123) are described in Materials and Methods. These cells were detached and incubated with biotin-labeled monoclonal antibody WM23 (against GPIbα) at 22°C for 30 min, and then incubated with phycoerythrin-labeled streptavidin and FITC-labeled monoclonal antibody D57 (against integrin αIIbβ3) at 22°C for 30 min. The cells were then analyzed by flow cytometry. (B) The cells described in A were incubated in vWF-coated microtiter wells for 30 min at 37°C in the presence of 5 μg/ml botrocetin. Untransfected CHO cells were also used as a negative control. To compare the function of αIIbβ3 expressed in 123 and 2b3a cells, cells are also incubated with fibrinogen (Fg)-coated wells. After washing, the adherent cells were quantitated by an acid phosphatase assay as described in Materials and Methods. Shown in the figure are the results from triplicate samples (mean ± SD). (C) 123 cells, 2b3a and CHO cells were also allowed to adhere to (10 μg/ml) vWF-coated microtiter wells in the absence of botrocetin. 123 cells were also preincubated with control IgG, or the monoclonal antibodies, AK2 and SZ2 against the vWF-binding site of GPIbα, and then allowed to adhere to vWF-coated wells in the absence of botrocetin. Note that AK2 and SZ2 inhibited cell adhesion to vWF.

Mentions: To analyze the roles of GPIb-IX and integrin αIIbβ3 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 αIIbβ3 (2b3a cells). A stable cell line was also established that expressed both GPIb-IX and integrin αIIbβ3 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 (Andrews et al. 1989). As a positive control, these cells were also incubated in fibrinogen-coated microtiter wells. Adherent cells were quantitated with an acid-phosphatase assay. As shown in Fig. 1, <10% of the 2b3a cells (expressing only αIIbβ3) 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 αIIbβ3 expressed in CHO cells (O'Toole et al. 1990), and is also consistent with results obtained in platelets showing that integrin αIIbβ3 interacts poorly with vWF without prior activation (Savage et al. 1992). The possibility of defective integrin function and expression in 2b3a cells can be excluded, as both the 2b3a cells and 123 cells but not 1b9 cells adhered to immobilized fibrinogen which is known to interact with integrin αIIbβ3 without prior activation (Coller 1980; Savage et al. 1995; Fig. 1 B). Thus, vWF is a poor ligand for unactivated integrin αIIbβ3.


Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

Gu M, Xi X, Englund GD, Berndt MC, Du X - J. Cell Biol. (1999)

Adhesion of cells expressing recombinant receptors for vWF. (A) Cells expressing integrin αIIbβ3 (2b3a), cells expressing GPIb-IX (1b9) and cells expressing both GPIb-IX and αIIbβ3 (123) are described in Materials and Methods. These cells were detached and incubated with biotin-labeled monoclonal antibody WM23 (against GPIbα) at 22°C for 30 min, and then incubated with phycoerythrin-labeled streptavidin and FITC-labeled monoclonal antibody D57 (against integrin αIIbβ3) at 22°C for 30 min. The cells were then analyzed by flow cytometry. (B) The cells described in A were incubated in vWF-coated microtiter wells for 30 min at 37°C in the presence of 5 μg/ml botrocetin. Untransfected CHO cells were also used as a negative control. To compare the function of αIIbβ3 expressed in 123 and 2b3a cells, cells are also incubated with fibrinogen (Fg)-coated wells. After washing, the adherent cells were quantitated by an acid phosphatase assay as described in Materials and Methods. Shown in the figure are the results from triplicate samples (mean ± SD). (C) 123 cells, 2b3a and CHO cells were also allowed to adhere to (10 μg/ml) vWF-coated microtiter wells in the absence of botrocetin. 123 cells were also preincubated with control IgG, or the monoclonal antibodies, AK2 and SZ2 against the vWF-binding site of GPIbα, and then allowed to adhere to vWF-coated wells in the absence of botrocetin. Note that AK2 and SZ2 inhibited cell adhesion to vWF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169335&req=5

Figure 1: Adhesion of cells expressing recombinant receptors for vWF. (A) Cells expressing integrin αIIbβ3 (2b3a), cells expressing GPIb-IX (1b9) and cells expressing both GPIb-IX and αIIbβ3 (123) are described in Materials and Methods. These cells were detached and incubated with biotin-labeled monoclonal antibody WM23 (against GPIbα) at 22°C for 30 min, and then incubated with phycoerythrin-labeled streptavidin and FITC-labeled monoclonal antibody D57 (against integrin αIIbβ3) at 22°C for 30 min. The cells were then analyzed by flow cytometry. (B) The cells described in A were incubated in vWF-coated microtiter wells for 30 min at 37°C in the presence of 5 μg/ml botrocetin. Untransfected CHO cells were also used as a negative control. To compare the function of αIIbβ3 expressed in 123 and 2b3a cells, cells are also incubated with fibrinogen (Fg)-coated wells. After washing, the adherent cells were quantitated by an acid phosphatase assay as described in Materials and Methods. Shown in the figure are the results from triplicate samples (mean ± SD). (C) 123 cells, 2b3a and CHO cells were also allowed to adhere to (10 μg/ml) vWF-coated microtiter wells in the absence of botrocetin. 123 cells were also preincubated with control IgG, or the monoclonal antibodies, AK2 and SZ2 against the vWF-binding site of GPIbα, and then allowed to adhere to vWF-coated wells in the absence of botrocetin. Note that AK2 and SZ2 inhibited cell adhesion to vWF.
Mentions: To analyze the roles of GPIb-IX and integrin αIIbβ3 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 αIIbβ3 (2b3a cells). A stable cell line was also established that expressed both GPIb-IX and integrin αIIbβ3 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 (Andrews et al. 1989). As a positive control, these cells were also incubated in fibrinogen-coated microtiter wells. Adherent cells were quantitated with an acid-phosphatase assay. As shown in Fig. 1, <10% of the 2b3a cells (expressing only αIIbβ3) 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 αIIbβ3 expressed in CHO cells (O'Toole et al. 1990), and is also consistent with results obtained in platelets showing that integrin αIIbβ3 interacts poorly with vWF without prior activation (Savage et al. 1992). The possibility of defective integrin function and expression in 2b3a cells can be excluded, as both the 2b3a cells and 123 cells but not 1b9 cells adhered to immobilized fibrinogen which is known to interact with integrin αIIbβ3 without prior activation (Coller 1980; Savage et al. 1995; Fig. 1 B). Thus, vWF is a poor ligand for unactivated integrin αIIbβ3.

Bottom Line: 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 GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3).Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ilinois 60612, USA.

ABSTRACT
We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) 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 GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

Show MeSH
Related in: MedlinePlus