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 (PGI(2)) 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 PGI(2), 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 PGI(2). The effect of PGI(2) is mediated through decreased phosphorylation of CLEC-2, Syk and PLC gamma 2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC-2 antibody and LECs is not affected by PGI(2) and GSNO. Consistent with this, CLEC-2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI(2). Conclusions The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI(2) and NO.
CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP / Alessandra, Borgognone; Leyre Navarro, Nunez; Joao Natalino, Correia; Alice Y., Pollitt; G., Thomas Steven; Johannes A., Eble; Pulcinelli, FABIO MARIA; Melanie, Madhani; Steve P., Watson. - In: JOURNAL OF THROMBOSIS AND HAEMOSTASIS. - ISSN 1538-7933. - STAMPA. - 12:4(2014), pp. 550-559. [10.1111/jth.12514]
CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP
PULCINELLI, FABIO MARIA;
2014
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 (PGI(2)) 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 PGI(2), 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 PGI(2). The effect of PGI(2) is mediated through decreased phosphorylation of CLEC-2, Syk and PLC gamma 2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC-2 antibody and LECs is not affected by PGI(2) and GSNO. Consistent with this, CLEC-2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI(2). Conclusions The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI(2) and NO.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.