PMC

The adaptor protein Grb-2-associated binding protein-1 (Gab1) associates with Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) and phosphatidylinositol 3-kinase on platelet activation. These associations are enhanced in the absence of platelet endothelial cell adhesion molecule-1 (PECAM-1). Gab1 was immunoprecipitated from washed human platelets and platelets derived from PECAM-1-deficient and wild-type (WT) mice following stimulation of PECAM-1 signaling by antibody crosslinking (XL) (A, B) or collagen (C–F) for 90 s. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotted to detect SHP-2 (A, C, E) and p85 (B, D, F). Numerical data represent the percentage change of Gab1–SHP-2 or Gab1–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05. IP, immunoprecipitation.

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.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates the association of p85 with SHP-2. Washed human platelets (A, B) and platelets derived from PECAM-1-deficient and wild-type (WT) mice (D) were treated with EGTA (1 mm), apyrase (2 U mL⁻¹) and indomethacin (10 μm) prior to PECAM-1 stimulation by antibody crosslinking (XL) (A) or stimulation with collagen for 90 s (B, D). The levels of p85 associated with SHP-2 were detected before equivalent protein loading was verified by reprobing for Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2). (C) Far-western blotting for SHP-2–p85 interaction was performed on lysates of cells stimulated with collagen (25 μg mL⁻¹) for 90 s, resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to poly(vinylidine difluoride) membranes. The membranes were incubated with glutathione-S-transferase (GST) fusion protein containing the N-terminal SH2 domain of p85 (GST–p85-N-SH2) or GST control, followed by anti-GST and secondary antibodies. Blots were washed and incubated for 2 h with horseradish peroxidase-conjugated anti-goat IgG antibody (1 : 8000), and signals were detected by chemifluorescence. Numerical data represent the percentage change of SHP-2–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: **P ≤ 0.01 and ***P ≤ 0.001. IP, immunoprecipitation.

Working model for the modulation of collagen-stimulated phosphatidylinositol 3-kinase (PI3K) signaling and platelet function by platelet endothelial cell adhesion molecule-1 (PECAM-1). Homophilic ligand binding or clustering of PECAM-1 or glycoprotein (GP)VI activation by collagen results in stimulation of tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motifs present in the cytoplasmic tail of PECAM-1. This results in the recruitment and activation of the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2). Collagen stimulation of platelets results in the formation of a complex between PI3K and the adaptor protein Grb-2-associated binding protein-1 (Gab1), which also binds to linker for activation of T cells (LAT), forming a signaling complex. SHP-2 is also capable of joining this complex, an interaction that is enhanced in the absence of PECAM-1 signaling. The stimulation of PECAM-1 results in the recruitment of p85 to bind to PECAM-1. The ability in vitro of SHP-2 to directly interact with p85 suggests that the interaction of p85 and PECAM-1 is mediated indirectly by the phosphatase. Indeed, the interaction between SHP-2 and p85 is dramatically reduced in the absence of PECAM-1, suggesting that PECAM-1 controls this interaction. Consistent with studies in another cell types where SHP-2 disrupts Gab1 and p85 interactions, through dephosphorylation of a tyrosine required for binding, the absence of PECAM-1 results in stabilization of the interaction between Gab1 and p85. This indicates that PECAM-1 signaling results in the loss of PI3K from the LAT signalosome and reduced levels of PI3K signaling. The relative redistribution of p85 from the LAT signalosome may be correlated with the inhibition of PI3K signaling. This provides a mechanism by which the activation of PECAM-1 results in negative feedback to activation pathways. GEM, glycolipid-enriched membrane; Syk, spleen tyrosine kinase.

The linker for activation of T cells (LAT) signalosome is modulated by platelet endothelial cell adhesion molecule-1 (PECAM-1). Washed human platelets and platelets derived from PECAM-1-deficient and wild-type (WT) mice were treated with EGTA (1 mm), apyrase (2 U mL⁻¹) and indomethacin (10 μm) prior to stimulation with collagen or collagen-related peptide (CRP) for 90 s, and for human platelets in the presence or absence of PECAM-1 activation by antibody crosslinking (XL). (A, C, D) Levels of p85 associated with LAT were detected before equivalent protein loading was verified by reprobing for LAT. (B) Levels of phospholipase C (PLC)γ2 phosphorylation were determined before equivalent protein loading was verified by reprobing for PLCγ2. (E) Human platelets were stimulated with the glycoprotein VI-specific agonist CRP, and the effects of prior stimulation of PECAM-1 signaling, through antibody-mediated crosslinking (PECAM-1 XL), was determined by Western blot analysis of whole cell extracts separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Phosphatidylinositol 3-kinase signaling was measured through assessment of protein kinase B (PKB)α/Akt phosphorylation (Ser473) by immunoblot analysis with a phosphospecific antibody. Equivalent protein loading was verified by reprobing for total PKB/Akt. Densitometry analysis was performed on replicate experiments, and data were normalized for total protein loading levels (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05 and **P ≤ 0.01.