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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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GPIb-IX–induced soluble fibrinogen binding to integrin αIIbβ3. (A) Cells expressing both GPIb-IX and integrin αIIbβ3 (123 cells) were incubated with FITC-labeled fibrinogen (15 μg/ml) and 1 mg/ml ristocetin in the absence (Fg) or presence of RGDS peptide (Fg+RGDS). (B) 123 cells were incubated with FITC-labeled fibrinogen, ristocetin, and 20 μg/ml purified human vWF in the absence of RGDS (Fg) or in the presence of RGDS (Fg+RGDS). (C) Control mouse IgG or a monoclonal antibody against GPIbα, AK2, were added to 123 cells. The cells were then incubated with FITC-labeled fibrinogen, ristocetin and vWF. Note that the increased binding of fibrinogen in the presence of vWF was inhibited by RGDS and by AK2. (D) 2b3a cells expressing αIIbβ3 only were incubated with FITC-labeled fibrinogen, vWF and ristocetin. Cells in A–D were analyzed for fibrinogen binding by flow cytometry. (E) 123 cells were incubated with FITC-labeled fibrinogen in the presence of ristocetin only (Control), ristocetin and RGDS (+RGDS), ristocetin and vWF (+vWF), or ristocetin, vWF and RGDS (+vWF+RGDS) at 22°C for 30 min and examined for fibrinogen binding as described in A. Activation of fibrinogen binding was quantitated and expressed as an activation index which is the ratio of the fluorescence intensity (Geo mean) of sample cells over the fluorescence intensity (Geo mean) of the control 123 cells (not stimulated with vWF). Shown in E are the results of 4 experiments (mean ± SD). Student's t test revealed that the difference between control and vWF-stimulated (+vWF) fibrinogen binding is highly significant (P < 0.001).
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Figure 3: GPIb-IX–induced soluble fibrinogen binding to integrin αIIbβ3. (A) Cells expressing both GPIb-IX and integrin αIIbβ3 (123 cells) were incubated with FITC-labeled fibrinogen (15 μg/ml) and 1 mg/ml ristocetin in the absence (Fg) or presence of RGDS peptide (Fg+RGDS). (B) 123 cells were incubated with FITC-labeled fibrinogen, ristocetin, and 20 μg/ml purified human vWF in the absence of RGDS (Fg) or in the presence of RGDS (Fg+RGDS). (C) Control mouse IgG or a monoclonal antibody against GPIbα, AK2, were added to 123 cells. The cells were then incubated with FITC-labeled fibrinogen, ristocetin and vWF. Note that the increased binding of fibrinogen in the presence of vWF was inhibited by RGDS and by AK2. (D) 2b3a cells expressing αIIbβ3 only were incubated with FITC-labeled fibrinogen, vWF and ristocetin. Cells in A–D were analyzed for fibrinogen binding by flow cytometry. (E) 123 cells were incubated with FITC-labeled fibrinogen in the presence of ristocetin only (Control), ristocetin and RGDS (+RGDS), ristocetin and vWF (+vWF), or ristocetin, vWF and RGDS (+vWF+RGDS) at 22°C for 30 min and examined for fibrinogen binding as described in A. Activation of fibrinogen binding was quantitated and expressed as an activation index which is the ratio of the fluorescence intensity (Geo mean) of sample cells over the fluorescence intensity (Geo mean) of the control 123 cells (not stimulated with vWF). Shown in E are the results of 4 experiments (mean ± SD). Student's t test revealed that the difference between control and vWF-stimulated (+vWF) fibrinogen binding is highly significant (P < 0.001).

Mentions: 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 αIIbβ3) 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 αIIbβ3 complex, and by anti-human β3 antibody SZ21 (Fig. 2 A). These data indicated that spreading was mainly mediated by integrin αIIbβ3 and that endogenous integrins were unlikely to play a major role. It is unlikely that coexpression of GPIb-IX with αIIbβ3 in the 123 cell line resulted in constitutively active integrin αIIbβ3, 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-IX–mediated 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.


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)

GPIb-IX–induced soluble fibrinogen binding to integrin αIIbβ3. (A) Cells expressing both GPIb-IX and integrin αIIbβ3 (123 cells) were incubated with FITC-labeled fibrinogen (15 μg/ml) and 1 mg/ml ristocetin in the absence (Fg) or presence of RGDS peptide (Fg+RGDS). (B) 123 cells were incubated with FITC-labeled fibrinogen, ristocetin, and 20 μg/ml purified human vWF in the absence of RGDS (Fg) or in the presence of RGDS (Fg+RGDS). (C) Control mouse IgG or a monoclonal antibody against GPIbα, AK2, were added to 123 cells. The cells were then incubated with FITC-labeled fibrinogen, ristocetin and vWF. Note that the increased binding of fibrinogen in the presence of vWF was inhibited by RGDS and by AK2. (D) 2b3a cells expressing αIIbβ3 only were incubated with FITC-labeled fibrinogen, vWF and ristocetin. Cells in A–D were analyzed for fibrinogen binding by flow cytometry. (E) 123 cells were incubated with FITC-labeled fibrinogen in the presence of ristocetin only (Control), ristocetin and RGDS (+RGDS), ristocetin and vWF (+vWF), or ristocetin, vWF and RGDS (+vWF+RGDS) at 22°C for 30 min and examined for fibrinogen binding as described in A. Activation of fibrinogen binding was quantitated and expressed as an activation index which is the ratio of the fluorescence intensity (Geo mean) of sample cells over the fluorescence intensity (Geo mean) of the control 123 cells (not stimulated with vWF). Shown in E are the results of 4 experiments (mean ± SD). Student's t test revealed that the difference between control and vWF-stimulated (+vWF) fibrinogen binding is highly significant (P < 0.001).
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Figure 3: GPIb-IX–induced soluble fibrinogen binding to integrin αIIbβ3. (A) Cells expressing both GPIb-IX and integrin αIIbβ3 (123 cells) were incubated with FITC-labeled fibrinogen (15 μg/ml) and 1 mg/ml ristocetin in the absence (Fg) or presence of RGDS peptide (Fg+RGDS). (B) 123 cells were incubated with FITC-labeled fibrinogen, ristocetin, and 20 μg/ml purified human vWF in the absence of RGDS (Fg) or in the presence of RGDS (Fg+RGDS). (C) Control mouse IgG or a monoclonal antibody against GPIbα, AK2, were added to 123 cells. The cells were then incubated with FITC-labeled fibrinogen, ristocetin and vWF. Note that the increased binding of fibrinogen in the presence of vWF was inhibited by RGDS and by AK2. (D) 2b3a cells expressing αIIbβ3 only were incubated with FITC-labeled fibrinogen, vWF and ristocetin. Cells in A–D were analyzed for fibrinogen binding by flow cytometry. (E) 123 cells were incubated with FITC-labeled fibrinogen in the presence of ristocetin only (Control), ristocetin and RGDS (+RGDS), ristocetin and vWF (+vWF), or ristocetin, vWF and RGDS (+vWF+RGDS) at 22°C for 30 min and examined for fibrinogen binding as described in A. Activation of fibrinogen binding was quantitated and expressed as an activation index which is the ratio of the fluorescence intensity (Geo mean) of sample cells over the fluorescence intensity (Geo mean) of the control 123 cells (not stimulated with vWF). Shown in E are the results of 4 experiments (mean ± SD). Student's t test revealed that the difference between control and vWF-stimulated (+vWF) fibrinogen binding is highly significant (P < 0.001).
Mentions: 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 αIIbβ3) 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 αIIbβ3 complex, and by anti-human β3 antibody SZ21 (Fig. 2 A). These data indicated that spreading was mainly mediated by integrin αIIbβ3 and that endogenous integrins were unlikely to play a major role. It is unlikely that coexpression of GPIb-IX with αIIbβ3 in the 123 cell line resulted in constitutively active integrin αIIbβ3, 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-IX–mediated 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.

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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Related in: MedlinePlus