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

Summary 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.


Lack of effect of SNP on CLEC-2-dependent platelet aggregation and tyrosine
phosphorylation-The effect of SNP (100 M) on CLEC-2 mAb-dependent platelet aggregation (a) and tyrosine phosphorylation (b) was studied. Washed platelets (2x10 8 /ml) were treated as indicated for 3 minutes prior to activation with mAb and allowed to aggregate (a). Washed platelets (4x10 8 /ml) treated with of apyrase (2 U/ml), indomethacin (10 M) and lotrafiban (10 M) were added with SNP (100 M) in comparison with PGI 2 (1 M) and stimulated with 10 g/ml CLEC-2 mAb for 3 minutes prior to lysis. Aliquots were analysed by SDS-PAGE and blots were probed with anti phospho-tyrosine monoclonal antibody (clone 4G10) (b).

Effect of cyclic nucleotide-elevation on CLEC-2-dependent platelet aggregation and tyrosine
phosphorylation in human platelets-The effect of PGI2 (0.1 M), GSNO and SNP (100 M) on rhodocytin-induced platelet aggregation (a) and tyrosine phosphorylation (b) was studied.

Rhodocytin-dependent tyrosine phosphorylation is partially reduced by cyclic nucleotide
reprobed for equal loading control. The total CLEC-2 blot was obtained by running in parallel 25% of the sample.

SUPPLEMENTARY FIGURE 5.
cAMP and cGMP have differential effects on different platelet pathways -Washed platelets (2×10 7 /ml) were pre-incubated for 3 min with the stated concentrations of PGI 2 , GSNO or vehicle and stimulated with 10 g/ml CLEC-2 mAb for 3 min (a) and 45 min ( 2x10 7 /ml washed platelets were treated with EHT1864 (50M), before spreading on podoplanin-coated coverslips (10g/ml). Coverslips were fixed, mounted and imaged as described in Figure 4 (c). A suspention of platelets (10 9 /ml) was activated by CLEC-2 mAb (10g/ml) for 45 min in the presence or absence of 1M PGI 2 and Rac activation was studied by GTP-Rac pull down assay using a commercially available kit according to manufacturer's instructions. A positive control for Rac activation was prepared by incubating platelets in parallel with GTPS as specified in the kit manual (not shown). Pulled down beads were analyzed by SDS-PAGE (10%) and blots were probed for Rac1(d) (n=3).
Washed platelets (4x10 8 /ml) were activated with 10 g/ml CLEC-2 mAb in the presence or absence of PGI 2 (1M) or GSNO (1mM) and lysed after 3, 20, 45 or 60 min with 5x Laemmli sample buffer. Aliquots were analysed by SDS-PAGE prior to probing with anti VASP p-Ser239. The antibody was then stripped and the membranes reprobed for VASP p-Ser157 and PLC2 for loading control (n=2).