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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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Defective thrombus formation in PTP-1B−/− mice. PTP-1B+/+ and PTP-1B−/− platelets were labeled ex vivo with Fura 2-AM and were reinfused into PTP-1B+/+ and PTP-1B−/− recipient mice, respectively. Then, vessel walls of arterioles in recipient cremaster muscles were subjected to laser injury, and the accumulation of fluorescent platelets into developing thrombi was assessed. (a) Representative composite brightfield and fluorescence images of Fura 2–labeled platelets up to 90 s after laser injury of an arteriole. Green represents labeled platelets and yellow represents cytoplasmic free calcium. Blood flow is from bottom to top. See Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1) for examples of thrombus formation in PTP-1B+/+ and PTP-1B−/− mice, respectively. (b) Accumulation of fluorescent platelets into the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 380 nm. FPlatelet is defined as the integrated fluorescence intensity associated with platelets. (c) Calcium mobilization within fluorescent platelets of the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 340 nm. FCalcium mobilization is defined as the integrated fluorescence intensity associated with calcium mobilization. Each curve in b and c is a composite of 18 independent thrombi generated in three mice (six thrombi per mouse). To analyze these data, all 18 curves were plotted versus time, and median values were determined at each time point and depicted in the figure.
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fig6: Defective thrombus formation in PTP-1B−/− mice. PTP-1B+/+ and PTP-1B−/− platelets were labeled ex vivo with Fura 2-AM and were reinfused into PTP-1B+/+ and PTP-1B−/− recipient mice, respectively. Then, vessel walls of arterioles in recipient cremaster muscles were subjected to laser injury, and the accumulation of fluorescent platelets into developing thrombi was assessed. (a) Representative composite brightfield and fluorescence images of Fura 2–labeled platelets up to 90 s after laser injury of an arteriole. Green represents labeled platelets and yellow represents cytoplasmic free calcium. Blood flow is from bottom to top. See Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1) for examples of thrombus formation in PTP-1B+/+ and PTP-1B−/− mice, respectively. (b) Accumulation of fluorescent platelets into the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 380 nm. FPlatelet is defined as the integrated fluorescence intensity associated with platelets. (c) Calcium mobilization within fluorescent platelets of the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 340 nm. FCalcium mobilization is defined as the integrated fluorescence intensity associated with calcium mobilization. Each curve in b and c is a composite of 18 independent thrombi generated in three mice (six thrombi per mouse). To analyze these data, all 18 curves were plotted versus time, and median values were determined at each time point and depicted in the figure.

Mentions: Thrombus formation can be studied in living mice by real-time fluorescence and brightfield microscopy of cremaster muscle arterioles that were subjected to laser injury (Falati et al., 2002). Platelets from PTP-1B−/− and PTP-1B+/+ mice were labeled with the Ca2+-sensitive fluorescent dye Fura 2 and were reinfused into PTP-1B−/− and PTP-1B+/+ mice, respectively. Labeled donor platelets accounted for ∼20% of total platelets in the recipients. This enabled quantification of fluorescent platelet accumulation and mobilization of intracellular Ca2+ in developing thrombi at sites of laser injury (Fig. 6 and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1). As described previously for other normal mouse platelets (Falati et al., 2002), PTP-1B+/+ platelets accumulated into a growing thrombus for 60–120 s, and some platelets detached over the course of several minutes. Platelet calcium mobilization increased over roughly the same time course. In contrast, the quantity of PTP-1B−/− platelets that incorporated into a growing thrombus was markedly reduced, and those platelets that did become incorporated tended to detach rapidly and exhibited little calcium mobilization (Fig. 6 and Videos 1 and 2). Similar results were obtained when labeled PTP-1B−/− platelets were reinfused into PTP-1B+/+ mice, indicating that the defect was intrinsic to PTP-1B−/− platelets (17 thrombi were analyzed in three PTP-1B+/+ mice; not depicted). Thus, in this model of vascular injury, PTP-1B is required for calcium mobilization and stable platelet accumulation into growing thrombi.


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)

Defective thrombus formation in PTP-1B−/− mice. PTP-1B+/+ and PTP-1B−/− platelets were labeled ex vivo with Fura 2-AM and were reinfused into PTP-1B+/+ and PTP-1B−/− recipient mice, respectively. Then, vessel walls of arterioles in recipient cremaster muscles were subjected to laser injury, and the accumulation of fluorescent platelets into developing thrombi was assessed. (a) Representative composite brightfield and fluorescence images of Fura 2–labeled platelets up to 90 s after laser injury of an arteriole. Green represents labeled platelets and yellow represents cytoplasmic free calcium. Blood flow is from bottom to top. See Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1) for examples of thrombus formation in PTP-1B+/+ and PTP-1B−/− mice, respectively. (b) Accumulation of fluorescent platelets into the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 380 nm. FPlatelet is defined as the integrated fluorescence intensity associated with platelets. (c) Calcium mobilization within fluorescent platelets of the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 340 nm. FCalcium mobilization is defined as the integrated fluorescence intensity associated with calcium mobilization. Each curve in b and c is a composite of 18 independent thrombi generated in three mice (six thrombi per mouse). To analyze these data, all 18 curves were plotted versus time, and median values were determined at each time point and depicted in the figure.
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Related In: Results  -  Collection

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fig6: Defective thrombus formation in PTP-1B−/− mice. PTP-1B+/+ and PTP-1B−/− platelets were labeled ex vivo with Fura 2-AM and were reinfused into PTP-1B+/+ and PTP-1B−/− recipient mice, respectively. Then, vessel walls of arterioles in recipient cremaster muscles were subjected to laser injury, and the accumulation of fluorescent platelets into developing thrombi was assessed. (a) Representative composite brightfield and fluorescence images of Fura 2–labeled platelets up to 90 s after laser injury of an arteriole. Green represents labeled platelets and yellow represents cytoplasmic free calcium. Blood flow is from bottom to top. See Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1) for examples of thrombus formation in PTP-1B+/+ and PTP-1B−/− mice, respectively. (b) Accumulation of fluorescent platelets into the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 380 nm. FPlatelet is defined as the integrated fluorescence intensity associated with platelets. (c) Calcium mobilization within fluorescent platelets of the developing thrombus. Fluorescent signal was detected at 510 nm after excitation at 340 nm. FCalcium mobilization is defined as the integrated fluorescence intensity associated with calcium mobilization. Each curve in b and c is a composite of 18 independent thrombi generated in three mice (six thrombi per mouse). To analyze these data, all 18 curves were plotted versus time, and median values were determined at each time point and depicted in the figure.
Mentions: Thrombus formation can be studied in living mice by real-time fluorescence and brightfield microscopy of cremaster muscle arterioles that were subjected to laser injury (Falati et al., 2002). Platelets from PTP-1B−/− and PTP-1B+/+ mice were labeled with the Ca2+-sensitive fluorescent dye Fura 2 and were reinfused into PTP-1B−/− and PTP-1B+/+ mice, respectively. Labeled donor platelets accounted for ∼20% of total platelets in the recipients. This enabled quantification of fluorescent platelet accumulation and mobilization of intracellular Ca2+ in developing thrombi at sites of laser injury (Fig. 6 and Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1). As described previously for other normal mouse platelets (Falati et al., 2002), PTP-1B+/+ platelets accumulated into a growing thrombus for 60–120 s, and some platelets detached over the course of several minutes. Platelet calcium mobilization increased over roughly the same time course. In contrast, the quantity of PTP-1B−/− platelets that incorporated into a growing thrombus was markedly reduced, and those platelets that did become incorporated tended to detach rapidly and exhibited little calcium mobilization (Fig. 6 and Videos 1 and 2). Similar results were obtained when labeled PTP-1B−/− platelets were reinfused into PTP-1B+/+ mice, indicating that the defect was intrinsic to PTP-1B−/− platelets (17 thrombi were analyzed in three PTP-1B+/+ mice; not depicted). Thus, in this model of vascular injury, PTP-1B is required for calcium mobilization and stable platelet accumulation into growing thrombi.

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