CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP.

The C‐type lectin receptor CLEC‐2 has a single YxxL (hemITAM) in its cytoplasmic tail, which is phosphorylated upon ligand engagement by the interplay of Src and Syk tyrosine kinases 1. In turn, Syk is recruited via its tandem SH2 domains to two phosphorylated CLEC‐2 tails, leading to initiation of a downstream signaling cascade involving LAT, SLP‐76, PI3 kinase and PLCγ2, which culminates in platelet activation 2. The only established physiological ligand for CLEC‐2 is podoplanin, a sialomucin‐like glycoprotein expressed in a variety of cells, including lymphatic endothelial cells (LECs). Several groups have shown that deletion of the gene encoding CLEC‐2, Clec1b, results in perinatal lethality in association with defects in lymphatic development 3 and a failure to inflate the lungs at birth 5. PF4‐Cre Clec1bfl/fl transgenic mice, which are deficient of CLEC‐2 in the platelet/megakaryocyte lineage, also have blood‐filled lymphatics 5. The same phenotype is seen following deletion of the CLEC‐2 signaling proteins, Syk, SLP‐76 and PLCγ2 4, or its ligand, podoplanin 4. These observations indicate that the activation of CLEC‐2 on platelets by podoplanin is necessary for prevention of blood‐lymphatic mixing during embryonic development 5. Blood‐filled lymphatics are found in the intestines of radiation chimeric mice reconstituted with Clec1b−/− fetal liver cells, indicating that CLEC‐2 is also necessary to repair the integrity of the intestinal lymphatic system. The interaction between platelet CLEC‐2 and podoplanin expressed on reticular fibroblastic cells is important in high endothelial venules, where it maintains integrity during immune responses 8. The cyclic nucleotide‐elevating agents, NO and PGI2, are released by endothelial cells in the vasculature 9. LECs generate prostanoids, including PGI2 10, as well as NO 12, and these have been shown to modulate the contractile activity of the collecting lymphatics 13. The observation that podoplanin and CLEC‐2 are critical for prevention of blood‐lymphatic mixing during development raises the question of how podoplanin is able to activate CLEC‐2 in the presence of PGI2 and NO, and therefore elevated cAMP and cGMP, given the powerful inhibitory action of the two cyclic nucleotides 9. Herein, we show that platelet aggregation induced by rhodocytin, CLEC‐2 antibody or podoplanin is inhibited by PGI2 but not by NO‐donors, and that platelet spreading on CLEC‐2 agonists is relatively insensitive to cyclic nucleotide‐elevating agents. These observations have important implications for understanding the molecular basis of platelet regulation in the development of the lymphatic system in mice.