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.
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.
Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. email@example.comShow MeSH
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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.
Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. firstname.lastname@example.org