Abstract
The activation of platelet CLEC-2 by podoplanin on lymphatic endothelial cells (LECs) has a critical role in prevention of mixing of lymphatic and blood vasculatures during embryonic development. Paradoxically, LECs release cAMP and cGMP-elevating agents, prostacyclin (PGI2 ) and nitric oxide (NO), respectively, which are powerful inhibitors of platelet activation. This raises the question of how podoplanin is able to activate CLEC-2 in the presence of the inhibitory cyclic nucleotides. We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets. We used rhodocytin, CLEC-2 monoclonal antibody, LECs and recombinant podoplanin as CLEC-2 agonists on mouse platelets. The effects of the cyclic nucleotide-elevating agents PGI2 , forskolin and the NO-donor GSNO were assessed with light transmission aggregometry, flow cytometry, protein phosphorylation and fluorescent imaging of platelets on LECs. We show that platelet aggregation induced by CLEC-2 agonists is resistant to GSNO but inhibited by PGI2 . The effect of PGI2 is mediated through decreased phosphorylation of CLEC-2, Syk and PLCγ2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC-2 antibody and LECs is not affected by PGI2 and GSNO. Consistent with this, CLEC-2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI2 . The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO.
Highlights
We show that platelet aggregation induced by rhodocytin, CLEC-2 antibody or podoplanin is inhibited by PGI2 but not by nitric oxide (NO)-donors, and that platelet spreading on CLEC-2 agonists is relatively insensitive to cyclic nucleotide-elevating agents
We have shown that CLEC-2-dependent activation of human platelets is regulated by release of the secondary mediators ADP and TXA2 [20]
Apyrase and indomethacin had no significant effect on aggregation induced by rhodocytin, and caused a minor delay in response to 3 lg mLÀ1 CLEC-2 mAb (Figure S1)
Summary
We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets
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