The role of adenylyl cyclase 6 in platelet function and thrombosis

Platelet activation is 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 adenylyl cyclase (AC), protein kinase A (PKA) and phosphodiesterase’s (PDEs). AC6 is the predominant isoform in both human and murine platelets, although its importance to platelet function is unclear. To address this, we generated a novel platelet-specific AC6 deficient mouse (AC6-KO). This new murine strain was fertile and showed no differences in platelet counts or morphology. In vitro studies showed that the absence of AC6 compromised the ability of PGI2 to control thrombin-mediated aggregation, fibrinogen binding, P-selectin expression, and PS exposure. In contrast, there were no effects on inhibition of platelet activation stimulated by collagen. In vivo studies indicated an accelerated rate of thrombosis in response to ferric chloride injury, as well as impaired thrombus stability in the AC6-KO compared to controls. Having found that inhibition of thrombin-mediated platelet activation was compromised in the absence of AC6, we examined the role of AC6 in cAMP signalling. Interestingly, no differences in basal cAMP concentrations were 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. We found that phosphoVASPSer239, phosphoVASPSer157 and phosphoGSK3βSer9 were all significantly impaired in AC6-KO platelets in response to both PGI2 and forskolin, indicating that reduced cAMP led to diminished signalling responses. Overall, 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 platelet function and thrombosis is linked to multiple AC isoforms.