Investigating novel platelet immunoreceptor ‘CEACAM2’ in contact-dependent events that modulate platelet thrombus formation

CVD is the general term for a class of heart diseases involving heart and blood vessels. Whilst the term commonly refers to any form of disorder affecting the cardiovascular system, it is most typically linked to atherosclerosis. The mortality rates for CVD have increased in the past few years within high-income countries and it is now the leading cause of death in the Western world [5]. Thrombi are formed in response to damage to the endothelial lining of the blood vessels, which causes platelets to clump together and activate at the site of damage. In the pathological sequelae of atherosclerosis, rupture of collagen-rich plaques leads to in vivo platelet activation, adhesion and thrombus formation. In a physiological context, Ig-ITIM superfamily members negatively regulate the initial phase of contact-dependent events involving platelet-collagen GPVI interactions, thereby limiting the in vivo growth of thrombi. Few examples of naturally occurring inhibitors of platelet-collagen interactions have been described [6]. Prostacyclin and nitrous oxide (NO) are natural inhibitors released from the endothelium [7], while Ig-ITIM members appear to be the first identified natural inhibitors that serve an autoregulatory role when platelets come into contact with each other following exposure to collagen [8].

Carcinoembryonic antigen-related cell adhesion molecule-2 (CEACAM2) is a cell surface glycoprotein expressed on blood, epithelial and vascular cells. CEACAM2 possesses adhesive and signalling 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–/– deficient mice (Cc2–/–) revealed that CEACAM2 serves to negatively regulate collagen GPVI-FcRγ–chain and the C-type lectin-like receptor 2 (CLEC-2) signalling. Cc2–/– 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 hyper-responsive CRP and CLEC-2-induced alpha and dense granule release compared to wild-type platelets. Furthermore, using intravital microscopy to ferric chloride (FeCl3) injured mesenteric arterioles and laser induced injury of cremaster muscle arterioles, we herein show that thrombi formed in Cc2–/– 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.

Previous studies have implicated that the Ig-ITIM superfamily member, CEACAM1 may regulate integrin function. As CEACAM2 is the twin of CEACAM1, it is possible that CEACAM2 may also have a physiologic role in integrin aIIbb3-mediated platelet function. In this project, we investigated the functional importance of CEACAM2 in murine platelets. Using Ceacam2-deficient mice, we showed that they have prolonged tail bleeding times and volume of blood lost. Cc2–/– platelets have moderate integrin aIIbb3 mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and type I collagen and delayed kinetics in retraction of fibrin clots in vitro. This functional integrin aIIbb3 defect could not be attributed to altered integrin aIIbb3 expression. Cc2–/– platelets displayed normal ‘inside-out’ signalling properties as demonstrated by normal agonist-induced binding of soluble FITC-fibrinogen and JON/A antibody binding. This project provides direct evidence that CEACAM2 is essential for normal integrin aIIbb3-mediated platelet function and that disruption of mouse CEACAM2 induced a moderate integrin aIIbb3-mediated platelet functional defects.