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PTP-1B is an essential positive regulator of platelet integrin signaling.

Arias-Salgado EG, Haj F, Dubois C, Moran B, Kasirer-Friede A, Furie BC, Furie B, Neel BG, Shattil SJ - J. Cell Biol. (2005)

Bottom Line: In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process.Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B.Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.

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PTP-1B−/− platelets are defective in αIIbβ3-dependent spreading on fibrinogen. (a) Platelets from PTP-1B+/+ and PTP-1B−/− mice were plated on fibrinogen-coated coverslips for 40 min at RT in the presence or absence of 100 μM ADP. Adherent cells were fixed, permeabilized, and stained with rhodamine-phalloidin (F-actin, red) and antiphosphotyrosine antibodies (green). Images were acquired with a confocal fluorescence microscope. Bar, 10 μm. (b) Platelet surface areas from at least 25 images were analyzed and depicted in the left panel as means ± SEM. The right panel depicts the percentage of platelets containing one or more filopodia and/or lamellipodia. At least 80 cells each were analyzed.
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fig4: PTP-1B−/− platelets are defective in αIIbβ3-dependent spreading on fibrinogen. (a) Platelets from PTP-1B+/+ and PTP-1B−/− mice were plated on fibrinogen-coated coverslips for 40 min at RT in the presence or absence of 100 μM ADP. Adherent cells were fixed, permeabilized, and stained with rhodamine-phalloidin (F-actin, red) and antiphosphotyrosine antibodies (green). Images were acquired with a confocal fluorescence microscope. Bar, 10 μm. (b) Platelet surface areas from at least 25 images were analyzed and depicted in the left panel as means ± SEM. The right panel depicts the percentage of platelets containing one or more filopodia and/or lamellipodia. At least 80 cells each were analyzed.

Mentions: Outside-in signaling via αIIbβ3 facilitates platelet spreading on fibrinogen and platelet thrombus formation under conditions of flow (Phillips et al., 2001; Nesbitt et al., 2002; Shattil and Newman, 2004). Therefore, these responses were compared in PTP-1B−/− and PTP-1B+/+ platelets. PTP-1B−/− platelets attached but failed to spread on fibrinogen over 45 min, whereas PTP-1B+/+ platelets exhibited cytoskeletal reorganization, filopodial and lamellipodial extensions, and varying degrees of spreading (Fig. 4 a, no agonist). When spreading was assessed by computer analysis of mean platelet areas and the percentage of platelets with filopodia or lamellipodia was quantified, the differences between PTP-1B−/− and PTP-1B+/+ platelets were statistically significant (P < 0.001; Fig. 4 b).


PTP-1B is an essential positive regulator of platelet integrin signaling.

Arias-Salgado EG, Haj F, Dubois C, Moran B, Kasirer-Friede A, Furie BC, Furie B, Neel BG, Shattil SJ - J. Cell Biol. (2005)

PTP-1B−/− platelets are defective in αIIbβ3-dependent spreading on fibrinogen. (a) Platelets from PTP-1B+/+ and PTP-1B−/− mice were plated on fibrinogen-coated coverslips for 40 min at RT in the presence or absence of 100 μM ADP. Adherent cells were fixed, permeabilized, and stained with rhodamine-phalloidin (F-actin, red) and antiphosphotyrosine antibodies (green). Images were acquired with a confocal fluorescence microscope. Bar, 10 μm. (b) Platelet surface areas from at least 25 images were analyzed and depicted in the left panel as means ± SEM. The right panel depicts the percentage of platelets containing one or more filopodia and/or lamellipodia. At least 80 cells each were analyzed.
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Related In: Results  -  Collection

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fig4: PTP-1B−/− platelets are defective in αIIbβ3-dependent spreading on fibrinogen. (a) Platelets from PTP-1B+/+ and PTP-1B−/− mice were plated on fibrinogen-coated coverslips for 40 min at RT in the presence or absence of 100 μM ADP. Adherent cells were fixed, permeabilized, and stained with rhodamine-phalloidin (F-actin, red) and antiphosphotyrosine antibodies (green). Images were acquired with a confocal fluorescence microscope. Bar, 10 μm. (b) Platelet surface areas from at least 25 images were analyzed and depicted in the left panel as means ± SEM. The right panel depicts the percentage of platelets containing one or more filopodia and/or lamellipodia. At least 80 cells each were analyzed.
Mentions: Outside-in signaling via αIIbβ3 facilitates platelet spreading on fibrinogen and platelet thrombus formation under conditions of flow (Phillips et al., 2001; Nesbitt et al., 2002; Shattil and Newman, 2004). Therefore, these responses were compared in PTP-1B−/− and PTP-1B+/+ platelets. PTP-1B−/− platelets attached but failed to spread on fibrinogen over 45 min, whereas PTP-1B+/+ platelets exhibited cytoskeletal reorganization, filopodial and lamellipodial extensions, and varying degrees of spreading (Fig. 4 a, no agonist). When spreading was assessed by computer analysis of mean platelet areas and the percentage of platelets with filopodia or lamellipodia was quantified, the differences between PTP-1B−/− and PTP-1B+/+ platelets were statistically significant (P < 0.001; Fig. 4 b).

Bottom Line: In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process.Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B.Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.

Show MeSH
Related in: MedlinePlus