LAUSR

The immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) bearing receptors, PECAM-1 and CEACAM1 have been shown net negative regulators of platelet-collagen interactions and hemi-ITAM signalling 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 (CLEC-2), protease activated receptor PAR-4, ADP purinergic receptors and thromboxane receptor TP A2 pathways. Additionally, 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 TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared to hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen related peptide or rhodocytin. Contrastingly, DKO platelets released increased amounts of P-selectin upon stimulation with PAR-4 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 alpha granule release via PAR-4/Gαq/PLC signalling without crosstalk with Src/Syk or G12/13 signalling pathways. This DKO model showed a significant increase in thrombus formation compared to the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared to Pecam-1-/-, Ceacam1-/- and WT platelets. PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signalling, platelet-collagen interactions and PAR-4 Gαq protein coupled signalling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.