OC 35.2 - The role of Adenylyl Cyclase 6 in platelet cyclic-AMP signalling

Background: Platelet activation and haemostasis are constrained by endothelial-derived prostacyclin (PGI2) acting through a cyclic adenosine-5’-monophosphate (cAMP) signalling pathway. Cyclic-AMP signalling in platelets involves a complex network of multiple isoforms of both adenylyl cyclase (AC) and protein kinase A (PKA).

Aims: In the present study, we characterised the role of AC6, the predominant isoform in both human and murine platelets, on platelet function.

Methods: A platelet specific AC6 deficient mouse (AC6-KO) was created using a Cre-recombinase approach. Light transmission aggregometry, flow cytometry and intravital microscopy were used to assess platelet function. Western Blotting and EIA techniques were also used to assess downstream cAMP signalling and cAMP production, respectively.

Results: No difference in basal cAMP concentrations was observed between AC6-KO and littermate controls, suggesting that AC6 does not control basal cAMP production. In contrast, AC6-KO showed significantly reduced responses to PGI2 induced cAMP generation, although this was not completely ablated. Consistent with this data, the phosphorylation of key PKA substrates phosphoVASPSer239, phosphoVASPSer157 and glycogen synthase kinase beta were impaired in AC6-KO platelets in response to both PGI2 and forskolin compared to controls. Having found that cAMP signalling was compromised in the absence of AC6, we examined its functional importance. In vitro studies showed that the absence of AC6 compromised the ability of PGI2 control thrombin, but not collagen activated platelets. The ability of PGI2 to inhibit thrombin-induced aggregation, fibrinogen binding and P-selectin expression was also impaired in AC6-KO platelets. In vivo studies indicated an accelerated rate of thrombosis in response to ferric chloride injury.

Conclusion(s): These data confirm a key role of AC6 in controlling cAMP-mediated platelet regulation in vitro and in vivo. However, given that the effects of PGI2 and cAMP signalling are not ablated, suggests that the regulation of haemostasis and thrombosis is linked to multiple AC isoforms.