• CEACAM2 is a novel platelet immunoreceptor.• CEACAM2 negatively regulates platelet-collagen interactions and thrombus growth and stability in vitro, in vivo and CLEC-2 pathways.Carcinoembryonic antigen-related cell adhesion molecule-2 (CEACAM2) is a cell-surface glycoprotein expressed on blood, epithelial, and vascular cells. CEACAM2 possesses adhesive and signaling properties mediated by immunoreceptor tyrosine-based inhibitory motifs. In this study, we demonstrate that CEACAM2 is expressed on the surface and in intracellular pools of platelets. Functional studies of platelets from Ceacam2) revealed that CEACAM2 serves to negatively regulate collagen glycoprotein VI (platelet) (GPVI)-FcRg-chain and the C-type lectinlike receptor 2 (CLEC-2) signaling. Cc2 2/2 platelets displayed enhanced GPVI and CLEC-2-selective ligands, collagen-related peptide (CRP), collagen, and rhodocytin (Rhod)-mediated platelet aggregation. They also exhibited increased adhesion on type I collagen, and hyperresponsive CRP and CLEC-2-induced a and dense granule release compared with wild-type platelets. Furthermore, using intravital microscopy to ferric chloride (FeCl 3 )-injured mesenteric arterioles and laser-induced injury of cremaster muscle arterioles, we herein show that thrombi formed in Cc2 2/2 mice were larger and more stable than wild-type controls in vivo. Thus, CEACAM2 is a novel platelet immunoreceptor that acts as a negative regulator of platelet GPVI-collagen interactions and of ITAM receptor CLEC-2 pathways. (Blood. 2014;124(15):2431-2441

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an Ig-ITIM superfamily member that regulates integrin αIIbβ3 function. We hypothesized that its twin protein, CEACAM2, exerts a similar physiologic role in murine platelets. CEACAM2-deficient mice (Cc2) displayed prolonged tail bleeding times and increased volume of blood loss. Cc2 platelets have moderate integrin αIIbβ3-mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and type I collagen and delayed kinetics in the retraction of fibrin clots in vitro. This functional integrin αIIbβ3 defect could not be attributed to altered integrin αIIbβ3 expression. Cc2 platelets displayed normal 'inside-out' signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen and JON/A antibody binding. This data provides direct evidence that disruption of CEACAM2 induces a moderate integrin αIIbβ3-mediated platelet function defect, and that CEACAM2 is essential to maintain a normal integrin αIIbβ3-mediated platelet function.

Previous studies have implicated that the Ig-ITIM superfamily member, CEACAM1 may regulate integrin function. While CEACAM1 has been demonstrated to play a role as an inhibitory co-receptor of ITAM-associated GPVI/FcR γ-chain signaling pathways in platelets, its physiologic role in integrin αIIbβ3-mediated platelet function is unclear. In this study, we investigate the functional importance of Ceacam1 in murine platelets. We show that CEACAM1 is constitutively associated with integrin αIIbβ3 in resting human and mouse platelets as demonstrated by co-immunoprecipitation studies. Using Ceacam1-deficient mice, we show that they have prolonged tail bleeding times and volume of blood lost that is corrected by reconstitution with platelet Ceacam1. Ceacam1(-/-) platelets have moderate integrin αIIbβ3 mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and failure to retract fibrin clots in vitro. This functional integrin αIIbβ3 defect could not be attributed to altered integrin αIIbβ3 expression. Ceacam1(-/-) platelets displayed normal "inside-out" signaling properties as demonstrated by normal agonist-induced binding of soluble (fluorescein isothiocyanate) FITC-fibrinogen, JON/A antibody binding, and increases in cytosolic free calcium levels. This study provides direct evidence that Ceacam1 is essential for normal integrin αIIbβ3-mediated platelet function and that disruption of mouse Ceacam1 induced moderate integrin αIIbβ3-mediated functional defects.