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Abstract
Background
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.
Objectives
We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets.
Methods
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.
Results
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.
Conclusions
The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO.
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.
Objectives
We investigated the influence of cyclic nucleotides on CLEC-2 signaling in platelets.
Methods
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.
Results
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.
Conclusions
The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO.
| Original language | English |
|---|---|
| Pages (from-to) | 550-559 |
| Number of pages | 10 |
| Journal | Journal of Thrombosis and Haemostasis |
| Volume | 12 |
| Issue number | 4 |
| Early online date | 11 Apr 2014 |
| DOIs | |
| Publication status | Published - Apr 2014 |
Keywords
- blood platelets
- C-type lectin
- cyclic nucleotides
- lymphangiogenesis
- platelet activation
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- 3 Finished
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The Early Events in Signalling by Platelet ITAM and ITAM-Like Receptors
1/08/09 → 31/03/16
Project: Research