Molecular mechanisms of immunoreceptors in platelets

Platelets play an important role in physiological and pathological thrombosis. Upon vessel injury, platelets are recruited from the circulation to the site of injury and form a thrombus plug in order to stop the bleeding. Their molecular machines allow them to quickly be recruited to the site of injury and to be switched from resting to active state. However, there are number of checkpoints to maximise an adapted response and prevent overblown effects. Normally, potent natural bioactive negative regulators of platelet activation released from healthy endothelium are prostacyclin and nitric oxide, however, when the endothelium is damaged or diseased, it limits the availability of bioactive prostacyclin and nitric oxide. In this situation, the naturally occurring immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors play a more substantive role as negative regulators of platelet activation pathways. It is not known whether these ITIM-bearing receptors collectively work in platelets to tightly control platelet activation and tethering in wherever and whenever a fine-tuned response is needed in the body. Moreover, the regulation of hemi-immunoreceptor tyrosine-based activation motif (hemITAM) signalling pathway by ITIM-containing receptors is supported by previous studies but whether there is a collective contribution is not known.  Also, the regulation of GPCR signalling by ITIM-containing receptors is highly controversial. Therefore, to address this issue, mice lacking the classical ITIM-bearing receptors, PECAM-1 and Ceacam1 was developed and thus to examine functional consequences for in vitro and in vivo thrombus formation, hemITAM-mediated-GPCR signalling and other pathways in platelets. In this study, Pecam1-/- mouse was crossed with Ceacam1-/- mouse to derive a double knockout mouse model lacking both Pecam-1 and Ceacam1. Different in vitro and in vivo approaches were used to examine functional consequences for in vitro and in vivo thrombus formation, hemITAM-mediated-GPCR signalling and other pathways in platelets. Also, to study the effect of Pecam-1-/-. Ceacam1-/-  in platelets activation by glycoprotein VI, C-type lectin-like receptor 2 (CLEC-2), protease activated receptor PAR-4, ADP purinergic receptors and TP thromboxane A2 pathways. DKO platelets responded normally to ADP purinergic receptors and TP thromboxane 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. In contrast, 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 PLC or Rho A kinase revealed that DKO platelets enhanced alpha granule release via PAR-4/G-alpha-q/PLC signalling without crosstalk with Src/Syk or G12/13 signalling pathways. The DKO model revealed 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. In conclusion, these findings further clarified how the classical ITIM-containing receptors, Pecam-1 and Ceacam1, work in concert to negatively regulate hem-ITAM signalling, platelet-collagen interactions and PAR-4 G-alpha-q protein coupled signalling pathways. Therefore, both Pecam-1 and Ceacam1 are required for the negative regulation of platelet activation and microvascular thrombosis in vivo.