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Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells.

Moraes LA, Barrett NE, Jones CI, Holbrook LM, Spyridon M, Sage T, Newman DK, Gibbins JM - J. Thromb. Haemost. (2010)

Bottom Line: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI-Fc receptor (FcR)γ-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT).An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses.PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk

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Stimulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) signaling results in recruitment of phosphatidylinositol 3-kinase (PI3K). Washed human platelets were incubated with antibody specific for PECAM-1 crosslinking (XL) or isotype control prior to stimulation with collagen-related peptide (0.5 μg mL−1) for 90 s (A), or wild-type and PECAM-1-deficient mouse platelets were stimulated with collagen (2.5 μg mL−1) (B) and aggregation was measured under constant stirring conditions at 37 °C. Washed human platelets were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to stimulation of PECAM-1 by antibody crosslinking (C, E) or with collagen (D, F) for 45, 90 and 180 s. (C, D) Levels of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) associated with PECAM-1 were detected before equivalent protein loading was verified by reprobing for PECAM-1. Levels of p85 subunit of PI3K associated with PECAM-1 detected after stimulation with glycoprotein VI agonist collagen (25 μg mL−1) (E) or antibody specific for PECAM-1 crosslinking (1 μg mL−1) (F). Equivalent protein loading was verified by reprobing for PECAM-1. Immunoblots were visualized by fluorescence imaging, quantified, and normalized for protein loading. Numerical data represent the percentage change of PECAM-1–SHP-2 association in stimulated samples as compared with control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. IP, immunoprecipitation.
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fig01: Stimulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) signaling results in recruitment of phosphatidylinositol 3-kinase (PI3K). Washed human platelets were incubated with antibody specific for PECAM-1 crosslinking (XL) or isotype control prior to stimulation with collagen-related peptide (0.5 μg mL−1) for 90 s (A), or wild-type and PECAM-1-deficient mouse platelets were stimulated with collagen (2.5 μg mL−1) (B) and aggregation was measured under constant stirring conditions at 37 °C. Washed human platelets were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to stimulation of PECAM-1 by antibody crosslinking (C, E) or with collagen (D, F) for 45, 90 and 180 s. (C, D) Levels of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) associated with PECAM-1 were detected before equivalent protein loading was verified by reprobing for PECAM-1. Levels of p85 subunit of PI3K associated with PECAM-1 detected after stimulation with glycoprotein VI agonist collagen (25 μg mL−1) (E) or antibody specific for PECAM-1 crosslinking (1 μg mL−1) (F). Equivalent protein loading was verified by reprobing for PECAM-1. Immunoblots were visualized by fluorescence imaging, quantified, and normalized for protein loading. Numerical data represent the percentage change of PECAM-1–SHP-2 association in stimulated samples as compared with control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. IP, immunoprecipitation.

Mentions: PECAM-1 tyrosine phosphorylation and subsequent activation of signaling molecules is stimulated following PECAM-1 clustering (antibody or homophilic ligation) or following platelet activation [6,7]. Phosphorylation of PECAM-1 is associated with the inhibition of platelet function (Fig. 1A,B), as well as secretion and adhesion responses [6,7,9]. The activation of PECAM-1 signaling is also stimulated downstream of platelet activation, and has been proposed to represent a negative feedback mechanism [6,7]. The association of PECAM-1 with SHP-2 has been described previously, and shown to be mediated by the SH2 domains of this phosphatase [23,24,28,29]. In order to determine the kinetics and extent of SHP-2 recruitment by PECAM-1 following crosslinking of PECAM-1 or GPVI stimulation with collagen, human platelets were stimulated for 45 s, 1 min 30 s and 3 min, in the presence of EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) to prevent aggregation and ensure the study of primary signaling events. The level of SHP-2 associated with immunoprecipitated PECAM-1 was measured by immunoblot analysis. The extent of association between PECAM-1 and SHP-2 was dependent on the duration of stimulation, and was proportional to the increase in the level of tyrosine phophorylation of SHP-2 (Fig. 1C,D; Fig. S2). Changes in tyrosine phosphorylation, SHP-2 binding and PECAM-1 binding were detected at early time points (detectable at 45 s in Fig. S2) and continued to rise for 3 min. Under the conditions used, similar kinetics for PECAM-1–SHP-2– interactions were observed for PECAM-1 crosslinking and stimulation of platelets with collagen. In subsequent experiments, a time point of 90 s was chosen to ensure that quantification of association could be reliably measured with this approach.


Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells.

Moraes LA, Barrett NE, Jones CI, Holbrook LM, Spyridon M, Sage T, Newman DK, Gibbins JM - J. Thromb. Haemost. (2010)

Stimulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) signaling results in recruitment of phosphatidylinositol 3-kinase (PI3K). Washed human platelets were incubated with antibody specific for PECAM-1 crosslinking (XL) or isotype control prior to stimulation with collagen-related peptide (0.5 μg mL−1) for 90 s (A), or wild-type and PECAM-1-deficient mouse platelets were stimulated with collagen (2.5 μg mL−1) (B) and aggregation was measured under constant stirring conditions at 37 °C. Washed human platelets were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to stimulation of PECAM-1 by antibody crosslinking (C, E) or with collagen (D, F) for 45, 90 and 180 s. (C, D) Levels of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) associated with PECAM-1 were detected before equivalent protein loading was verified by reprobing for PECAM-1. Levels of p85 subunit of PI3K associated with PECAM-1 detected after stimulation with glycoprotein VI agonist collagen (25 μg mL−1) (E) or antibody specific for PECAM-1 crosslinking (1 μg mL−1) (F). Equivalent protein loading was verified by reprobing for PECAM-1. Immunoblots were visualized by fluorescence imaging, quantified, and normalized for protein loading. Numerical data represent the percentage change of PECAM-1–SHP-2 association in stimulated samples as compared with control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. IP, immunoprecipitation.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3298659&req=5

fig01: Stimulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) signaling results in recruitment of phosphatidylinositol 3-kinase (PI3K). Washed human platelets were incubated with antibody specific for PECAM-1 crosslinking (XL) or isotype control prior to stimulation with collagen-related peptide (0.5 μg mL−1) for 90 s (A), or wild-type and PECAM-1-deficient mouse platelets were stimulated with collagen (2.5 μg mL−1) (B) and aggregation was measured under constant stirring conditions at 37 °C. Washed human platelets were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to stimulation of PECAM-1 by antibody crosslinking (C, E) or with collagen (D, F) for 45, 90 and 180 s. (C, D) Levels of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) associated with PECAM-1 were detected before equivalent protein loading was verified by reprobing for PECAM-1. Levels of p85 subunit of PI3K associated with PECAM-1 detected after stimulation with glycoprotein VI agonist collagen (25 μg mL−1) (E) or antibody specific for PECAM-1 crosslinking (1 μg mL−1) (F). Equivalent protein loading was verified by reprobing for PECAM-1. Immunoblots were visualized by fluorescence imaging, quantified, and normalized for protein loading. Numerical data represent the percentage change of PECAM-1–SHP-2 association in stimulated samples as compared with control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. IP, immunoprecipitation.
Mentions: PECAM-1 tyrosine phosphorylation and subsequent activation of signaling molecules is stimulated following PECAM-1 clustering (antibody or homophilic ligation) or following platelet activation [6,7]. Phosphorylation of PECAM-1 is associated with the inhibition of platelet function (Fig. 1A,B), as well as secretion and adhesion responses [6,7,9]. The activation of PECAM-1 signaling is also stimulated downstream of platelet activation, and has been proposed to represent a negative feedback mechanism [6,7]. The association of PECAM-1 with SHP-2 has been described previously, and shown to be mediated by the SH2 domains of this phosphatase [23,24,28,29]. In order to determine the kinetics and extent of SHP-2 recruitment by PECAM-1 following crosslinking of PECAM-1 or GPVI stimulation with collagen, human platelets were stimulated for 45 s, 1 min 30 s and 3 min, in the presence of EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) to prevent aggregation and ensure the study of primary signaling events. The level of SHP-2 associated with immunoprecipitated PECAM-1 was measured by immunoblot analysis. The extent of association between PECAM-1 and SHP-2 was dependent on the duration of stimulation, and was proportional to the increase in the level of tyrosine phophorylation of SHP-2 (Fig. 1C,D; Fig. S2). Changes in tyrosine phosphorylation, SHP-2 binding and PECAM-1 binding were detected at early time points (detectable at 45 s in Fig. S2) and continued to rise for 3 min. Under the conditions used, similar kinetics for PECAM-1–SHP-2– interactions were observed for PECAM-1 crosslinking and stimulation of platelets with collagen. In subsequent experiments, a time point of 90 s was chosen to ensure that quantification of association could be reliably measured with this approach.

Bottom Line: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI-Fc receptor (FcR)γ-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT).An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses.PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk

Show MeSH
Related in: MedlinePlus