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


OBJECTIVE
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.
Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK; Department of Experimental Medicine, 'Sapienza' University of Rome, Rome, Italy.
A
A
Borgognone
L
L
Navarro-Núñez
J N
JN
Correia
A Y
AY
Pollitt
S G
SG
Thomas
J A
JA
Eble
F M
FM
Pulcinelli
M
M
Madhani
S P
SP
Watson
D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D011464:Epoprostenol / Q000378:metabolism; D037181:Lectins, C-Type / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D014757:Viper Venoms / Q000378:metabolism; D000098853:Podoplanin
D002118:Calcium; D000242:Cyclic AMP; D006152:Cyclic GMP; D011464:Epoprostenol; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D009569:Nitric Oxide; D014757:Viper Venoms; C529518:CLEC-2 protein, mouse; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C112292:rhodocytin protein, Calloselasma rhodostoma
C-type lectin; blood platelets; cyclic nucleotides; lymphangiogenesis; platelet activation