Platelet Endothelial Cell Adhesion Molecule-1 Inhibits Platelet Response to Thrombin and von Willebrand Factor by Regulating the Internalization of Glycoprotein Ib via AKT/Glycogen Synthase Kinase-3/Dynamin and Integrin αIIbβ3

Objective-Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. Approach and Results-Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin alpha IIb beta 3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3x10(-7)-1x10(-5) mol/L) and GYPGQV (3x10(-6)-1x10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ib alpha (GPIb alpha) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIb alpha by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIb alpha was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of beta 3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3 beta Ser(9), but interestingly an increase in glycogen synthase kinase-3 alpha pSer(21). PECAM-1-mediated internalization of GPIb alpha was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. Conclusions-PECAM-1 mediates internalization of GPIb alpha in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and alpha IIb beta 3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.