ABSTRACT
The Ig-ITIM bearing receptors, PECAM-1 and CEACAM1, have been shown net negative regulators of platelet-collagen interactions and hemiITAM signaling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2, protease activated receptor (PAR4), ADP purinergic receptors, and thromboxane receptor (TP) A2 pathways. In addition, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and the TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared with hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen-related peptide or rhodocytin. In contrast, DKO platelets showed increased amounts of P-selectin exposure upon stimulation with PAR4 agonist peptide or thrombin but not Pecam-1-/- , Ceacam1-/- , or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced α-granule release via PAR4/Gαq/PLC signaling without crosstalk with Src/Syk or G12/13 signaling pathways. Severely delayed clot retraction in vitro was observed in DKO phenotype. The DKO model revealed a significant increase in thrombus formation compared with the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared with Pecam-1-/- , Ceacam1-/- , and WT platelets. This study demonstrates that PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signaling, platelet-collagen interactions, and PAR4 Gαq protein- coupled signaling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.