Figure 6.

PGI₂ induces a PKA signalling response in spread platelets. Platelets (2 × 10⁸/ml) were spread on 100 μg/ml fibrinogen for 25 minutes in the presence or absence of PKA inhibitors 100 μM RP-8CPT-cAMP (RP) and 2 μM KT5720 (KT), before being washed with PBS. (a) The platelets were then treated with tyrodes containing 10 nM PGI₂ with or without PKA inhibitors (100 μM RP-8CPT-cAMP and 2 μM KT5720), or 1 μM forskolin, or Y27632 (10 μM), for a further 10 minutes. The samples were then lysed with laemelli buffer before being western blotted for pVASP^ser159, pMLC^ser19, pRhoA^ser188, and GAPDH. Cropped gel Images are representative of at least three experiments (full length gels are illustrated in Supplementary Figure S6). (b–d) Densitometry for the western blots; pVASP^ser159, pMLC^ser19 and pRhoA^ser188 using GAPDH as the loading control. The ratios were standardised to the control. (e) Spreading the platelets as above, they were treated with tyrodes containing 10 nM PGI₂ with or without PKA inhibitors 100 μM RP-8CPT-cAMP and 2 μM KT5720 for a further 10 minutes. The samples were then lysed, before the addition of RhoA GTP beads. Samples were then western blotted for active RhoA and total RhoA. Cropped gel Images are representative of at least three experiments (full length gels are illustrated in Supplementary Figure S6). (f) Images of RhoA pull down were analysed for densitometry (full length gels are illustrated in Supplementary Figure S6). Analysis is an average of at least n = 3 experiments. p < 0.05.