LAIR-1 and PECAM-1 function via the same signaling pathway to inhibit GPVI-mediated platelet activation

Background Inhibition of platelet responsiveness is important for controlling thrombosis. It is well established that platelet endothelial cell adhesion molecule-1 (PECAM_-1) serves as a physiological negative regulator of platelet-collagen interactions. We recently demonstrated that leukocyte-associated immunoglobulin-like receptor-1 (LAIR_-1) is a negative regulator of platelet production and reactivity. It is however not known if LAIR_-1 and PECAM_-1 function in the same or different inhibitory pathways. Objectives In this study, we investigated the role of LAIR_-1 alongside PECAM-1 in megakaryocyte development and platelet production and determined the functional redundancy through characterization of a LAIR_-1/PECAM_-1 double knockout (DKO) mouse model. Methods LAIR_-1 and PECAM_-1 expression in megakaryocytes were evaluated by western blotting. Megakaryocyte ploidy and proplatelet formation were evaluated by flow cytometry and fluorescent microscopy. Platelet function and signalling were compared in wild-type, LAIR_-1 −/− , PECAM_-1 −/− and DKO mice using aggregometry, flow cytometry and western blotting. Thrombosis was evaluated using the FeCl ₃ carotid artery model. Results We show that LAIR-₁/PECAM_-1 DKO mice exhibit a 17% increase in platelet count. Bone marrow-derived megakaryocytes from all 3 mouse models had normal ploidy in vitro, suggesting that neither LAIR-1 nor PECAM-1 regulates megakaryocyte development. Furthermore, relative to wild-type platelets, platelets derived from LAIR-₁, PECAM_-1, and DKO mice were equally hyperresponsive to collagen in vitro, indicating that LAIR-1 and PECAM-1 participate in the same inhibitory pathway. Interestingly, DKO mice exhibited normal thrombus formation in vivo due to DKO mouse platelets lacking the enhanced Src family kinase activation previously shown in platelets from LAIR_-1-deficient mice. Conclusion Findings from this study reveal that LAIR_-1 and PECAM_-1 act to inhibit GPVI-mediated platelet activation via the same signaling pathway. Mice lacking LAIR-1 and PECAM-1 do not however exhibit an increase in thrombus formation despite minor increase in platelet count and reactivity to collagen. This study adds to the growing evidence that immunoreceptor tyrosine-based inhibition motif–containing receptors are important regulators of platelet count and function.