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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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Role of PTP-1B in the interaction of platelets with fibrinogen and fibrin. (a) Platelet adhesion. Washed platelets (1.5 × 106 in 50 μl incubation buffer) were incubated in fibrinogen-coated microtiter wells for 1 h at RT, and platelet adhesion was quantified. Platelets from at least four mice were used to generate duplicate points at each fibrinogen concentration. (b) Soluble fibrinogen binding. Platelets were incubated at RT for 20 min with 150 μg/ml FITC-fibrinogen in the presence or absence of ADP, convulxin, or PAR4 receptor–activating peptide (AYPGKF; Faruqi et al., 2000). Fibrinogen binding was analyzed by flow cytometry. Data are the means ± SEM of quadruplicate determinations from an experiment that was representative of three that were performed. (c) Fibrin clot retraction was assessed 2 h after the addition of thrombin and CaCl2 to platelet-rich plasma. Clot volumes, expressed as a percentage of the initial volume of platelet-rich plasma, were significantly greater in PTP-1B−/− than in PTP+/+ samples, indicating less clot retraction. Data represent means ± SEM of seven experiments.
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fig5: Role of PTP-1B in the interaction of platelets with fibrinogen and fibrin. (a) Platelet adhesion. Washed platelets (1.5 × 106 in 50 μl incubation buffer) were incubated in fibrinogen-coated microtiter wells for 1 h at RT, and platelet adhesion was quantified. Platelets from at least four mice were used to generate duplicate points at each fibrinogen concentration. (b) Soluble fibrinogen binding. Platelets were incubated at RT for 20 min with 150 μg/ml FITC-fibrinogen in the presence or absence of ADP, convulxin, or PAR4 receptor–activating peptide (AYPGKF; Faruqi et al., 2000). Fibrinogen binding was analyzed by flow cytometry. Data are the means ± SEM of quadruplicate determinations from an experiment that was representative of three that were performed. (c) Fibrin clot retraction was assessed 2 h after the addition of thrombin and CaCl2 to platelet-rich plasma. Clot volumes, expressed as a percentage of the initial volume of platelet-rich plasma, were significantly greater in PTP-1B−/− than in PTP+/+ samples, indicating less clot retraction. Data represent means ± SEM of seven experiments.

Mentions: The stimulation of platelets with a G protein–coupled receptor agonist such as ADP results in more rapid and uniform platelet spreading on fibrinogen when compared with cells incubated without agonist (Haimovich et al., 1993). Thus, in addition to αIIbβ3 signaling, costimulatory pathways are involved in full platelet spreading. In contrast to the spreading defect of untreated PTP-1B−/− platelets, costimulation with ADP resulted in uniform, full spreading (Fig. 4, a and b). PTP-1B−/− platelets adhered normally to fibrinogen (Fig. 5 a), and they bound soluble fibrinogen normally in response to either ADP, PAR4 receptor–activating peptide, or convulxin, which is a glycoprotein VI agonist (Fig. 5 b). In addition, stirred PTP-1B−/− platelets that were incubated with 1–10 μM ADP or 250 μM PAR4 receptor–activating peptide exhibited an initial rate and extent of aggregation that was equivalent to those of PTP-1B+/+ platelets (unpublished data). On the other hand, PTP-1B−/− platelets mediated less fibrin clot retraction than PTP-1B+/+ platelets (P < 0.05); this response is dependent, in part, on αIIbβ3-triggered changes in the actin cytoskeleton (Fig. 5 c; Phillips et al., 2001; Shattil and Newman, 2004). Collectively, these results indicate that PTP-1B is required for normal outside-in αIIbβ3 signaling in platelets. However, PTP-1B appears to be dispensable for agonist induction of soluble fibrinogen binding to αIIbβ3 and for ADP costimulation of platelet spreading.


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)

Role of PTP-1B in the interaction of platelets with fibrinogen and fibrin. (a) Platelet adhesion. Washed platelets (1.5 × 106 in 50 μl incubation buffer) were incubated in fibrinogen-coated microtiter wells for 1 h at RT, and platelet adhesion was quantified. Platelets from at least four mice were used to generate duplicate points at each fibrinogen concentration. (b) Soluble fibrinogen binding. Platelets were incubated at RT for 20 min with 150 μg/ml FITC-fibrinogen in the presence or absence of ADP, convulxin, or PAR4 receptor–activating peptide (AYPGKF; Faruqi et al., 2000). Fibrinogen binding was analyzed by flow cytometry. Data are the means ± SEM of quadruplicate determinations from an experiment that was representative of three that were performed. (c) Fibrin clot retraction was assessed 2 h after the addition of thrombin and CaCl2 to platelet-rich plasma. Clot volumes, expressed as a percentage of the initial volume of platelet-rich plasma, were significantly greater in PTP-1B−/− than in PTP+/+ samples, indicating less clot retraction. Data represent means ± SEM of seven experiments.
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Related In: Results  -  Collection

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fig5: Role of PTP-1B in the interaction of platelets with fibrinogen and fibrin. (a) Platelet adhesion. Washed platelets (1.5 × 106 in 50 μl incubation buffer) were incubated in fibrinogen-coated microtiter wells for 1 h at RT, and platelet adhesion was quantified. Platelets from at least four mice were used to generate duplicate points at each fibrinogen concentration. (b) Soluble fibrinogen binding. Platelets were incubated at RT for 20 min with 150 μg/ml FITC-fibrinogen in the presence or absence of ADP, convulxin, or PAR4 receptor–activating peptide (AYPGKF; Faruqi et al., 2000). Fibrinogen binding was analyzed by flow cytometry. Data are the means ± SEM of quadruplicate determinations from an experiment that was representative of three that were performed. (c) Fibrin clot retraction was assessed 2 h after the addition of thrombin and CaCl2 to platelet-rich plasma. Clot volumes, expressed as a percentage of the initial volume of platelet-rich plasma, were significantly greater in PTP-1B−/− than in PTP+/+ samples, indicating less clot retraction. Data represent means ± SEM of seven experiments.
Mentions: The stimulation of platelets with a G protein–coupled receptor agonist such as ADP results in more rapid and uniform platelet spreading on fibrinogen when compared with cells incubated without agonist (Haimovich et al., 1993). Thus, in addition to αIIbβ3 signaling, costimulatory pathways are involved in full platelet spreading. In contrast to the spreading defect of untreated PTP-1B−/− platelets, costimulation with ADP resulted in uniform, full spreading (Fig. 4, a and b). PTP-1B−/− platelets adhered normally to fibrinogen (Fig. 5 a), and they bound soluble fibrinogen normally in response to either ADP, PAR4 receptor–activating peptide, or convulxin, which is a glycoprotein VI agonist (Fig. 5 b). In addition, stirred PTP-1B−/− platelets that were incubated with 1–10 μM ADP or 250 μM PAR4 receptor–activating peptide exhibited an initial rate and extent of aggregation that was equivalent to those of PTP-1B+/+ platelets (unpublished data). On the other hand, PTP-1B−/− platelets mediated less fibrin clot retraction than PTP-1B+/+ platelets (P < 0.05); this response is dependent, in part, on αIIbβ3-triggered changes in the actin cytoskeleton (Fig. 5 c; Phillips et al., 2001; Shattil and Newman, 2004). Collectively, these results indicate that PTP-1B is required for normal outside-in αIIbβ3 signaling in platelets. However, PTP-1B appears to be dispensable for agonist induction of soluble fibrinogen binding to αIIbβ3 and for ADP costimulation of platelet spreading.

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