Prostacyclin enlists AC6 to stop the clot

Prostacyclin is a naturally occurring bioactive metabolite of arachidonic acid that plays key roles in platelet aggregation and vascular relaxation (Moncada et al Nature 1976[1]). The discovery of prostacyclin led to the awarding of the Nobel Prize in Physiology or Medicine in 1982 to John Vane. Prostacyclin receptor knock-out (IP−/−) mice[2, 3] and genetic deficiency of prostacyclin signaling in human subjects[4], have confirmed prostacyclin’s critical protective role against thrombosis, intimal hyperplasia and restenosis, atherosclerosis, and myocardial reperfusion injury. Diminished prostacyclin signaling due to endothelial dysfunction also likely contributed to the thrombovascular disease and high mortality associated with the COVID-19 pandemic[5]. Clinically, prostacyclin analogues are approved therapies for pulmonary hypertension where microvascular thrombosis and vascular constriction contribute to disease. Despite the critical role prostacyclin plays in many thrombovascular disease processes, aside from pulmonary hypertension, few prostacyclin analogues are used clinically, with issues surrounding drug stability, targeting, expense, and side effects. In order to develop more efficacious and targeted prostacyclin or pathway analogues, a better understanding of the signaling pathways, particularly in the targets cell (platelets and/or vascular smooth muscle cells), is essential.

The human prostacyclin receptor (hIP—International Union of Pharmacology Receptor classification[6]) is a seven transmembrane G-protein coupled receptor (GPCR)[7]. The canonical pathway for PGI₂ binding to hIP involves Gαs, adenylyl cyclase, and PKA and subsequent complex intracellular effectors;[8] however, the key subtypes of effector pathway signaling protein intermediates in platelets remain unknown. In this issue of the Journal of Thrombosis and Haemostasis, Webb et al.[9] provide important studies to fill this knowledge gap, identifying adenylyl cyclase type 6 (AC6) as a key hIP-mediated signaling protein that mediates platelet inhibition. Specific deletion of platelet AC6 in a mouse model reduced platelet cAMP accumulation in response to prostacyclin analogue and reduced downstream signaling including the phosphorylation of PKA and PKA substrates. This decrease in prostacyclin signaling prevented the inhibitory effects of prostacyclin on P-selectin expression prostacyclin’s opposition of thrombin-induced fibrinogen binding, leading to pathological consequences of reduced bleeding time and increased arterial thrombosis in the AC6 platelet knockouts.

Webb and coworkers identify AC6 as just one of the cAMP generating enzymes in platelets[9]. Interestingly, AC6 knockdown did not alter basal cAMP, suggesting that other ACs are predominant contributors to basal cAMP and inhibition of platelet aggregation in the absence of prostacyclin. The fact that knockout of AC6 diminished only ~20% of forskolin-stimulated cAMP production is further evidence that several other AC isoforms are functional in these cells. These investigators detected AC5 and AC9 mRNA expression in addition to AC6. Each of the nine AC isoforms has distinct regulatory properties[10]. While all are stimulated by Gαs, only some are inhibited by Gi. Many other signaling moieties, including Gβγ, calcium-calmodulin, protein kinases, and nitric oxide, act on specific AC isoforms to influence AC activity[11]. AC6 is unique in that its activity is inhibited by virtually all of these signaling proteins, leading it to have low basal activity[12]. This makes it an appropriate signaling partner for GPCRs that need rapid and distinct signaling that leads to dynamic and specific control of cell function.

IP receptors are known to prefer coupling to AC6 in other cells and tissues[13–15]. Compartmentation of receptors and AC isoforms in membrane microdomains have been well described and likely underlie this preference of IP receptors for AC6 [11]. While GPRC-AC coupling is highly specific in some cells, Webb and coworkers show that higher PGI₂ concentrations can restore signaling and function[9]. This may result from less stringent compartmentation of cAMP signal generation in platelets or could be due to PGI₂ activation of other prostanoid receptors, such as EP2 and EP4 (which stimulate cAMP primarily through other AC isoforms[16]) at these higher concentrations. Regardless, Webb et. al.[9] demonstrate that IP receptors have a strong dependence on AC6 for both generation of, and cellular response to, cAMP. AC6 plays this outsized role in platelet function despite other AC isoforms being expressed. Of particular interest will be understanding the functional role of AC9 in platelets, as this isoform is the most poorly understood of the ACs[17].

The effects of loss or reduction platelet cAMP are clinically very important. The world-wide withdrawal of selective COX-2 inhibitors, rofecoxib (Vioxx^™) and valdecoxib (Bextra^™), was due to their discriminating suppression of COX-2-derived prostacyclin (PGI₂). While COX2 inhibition reduces pain-associated cytokines, the reduction in endothelial-derived prostacyclin led to increased risk of thrombosis and major adverse cardiovascular events (e.g., myocardial infarction and thrombotic stroke), particularly in patients with increased risk factors for cardiovascular disease[18]. The commonly used NSAIDs, including ibuprofen, can inhibit COX-1-derived thromboxane and COX-2-derived prostacyclin to different degrees, reducing or increasing thrombosis depending on the selectivity. This is very relevant particularly in patients with cardiovascular risk factors or existing disease where reduction of prostacyclin signaling can promote major adverse thrombovascular events. Moreover, over 150 genetic variants have been detected in and around the AC6 gene (ADCY6) (ADCY6[gene] - ClinVar - NCBI). The importance and relevance to thrombosis and hemostasis remain to be explored. Increasing cAMP through AC6 or other ACs isoforms could be an important therapeutic goal. Moreover, it remains of interest and importance to determine whether the AC6 isoform cross talks with the thromboxane pathway, particularly downstream phosphorylation events given the involvement of PKA and PKC. Prostacyclin signaling through hIP also promotes vasodilation and protective effects on vascular smooth muscle cell phenotype[19] and contributes to priming myometrial smooth muscle for the onset of labor[20]. Understanding specific AC signaling isoforms in other PGI₂-responsive tissues could have important implications for other facets of health in addition to this newly identified critical role in platelet prostacyclin signaling.