The critical role of platelet adenylyl cyclase 6 in hemostasis and thrombosis

Abstract

Background: Platelet activation is constrained by endothelial-derived prostacyclin (PGI₂) through cyclic adenosine-3',5'-monophosphate (cAMP) signaling involving multiple isoforms of adenylyl cyclase (AC). The roles of specific AC isoforms in controlling hemostasis remain unclear and require clarification.

Objectives: To understand the specific contribution of AC6 in platelet hemostatic and thrombotic function.

Methods: A platelet-specific AC6 knockout mouse was generated. Biochemical approaches were used to determine intracellular signaling, with flow cytometry, tail bleeding time assays, and in vivo thrombosis by ferric chloride were used to measure the hemostatic and thrombotic importance of platelet AC6.

Results: Loss of AC6 resulted in diminished accumulation of platelet cAMP in response to PGI_2, while basal cAMP was unaffected. We found no differences in phosphodiesterase 3A activity, suggesting the defect was in generation rather than hydrolysis of cAMP. Consistent with this, phosphorylation of protein kinase A substrates, vasodilator-stimulated phosphoprotein, and glycogen synthase kinase were diminished but not ablated. Functional studies demonstrated that the inhibition of thrombin-induced fibrinogen binding and P-selectin expression by PGI₂ was severely compromised, while inhibition of glycoprotein VI-mediated platelet activation was largely unaffected. Under conditions of flow formed stable thrombi, but in the absence of AC6, thrombi were insensitive to PGI₂. Diminished in vivo sensitivity to PGI₂ manifested as significantly reduced tail bleeding and accelerated occlusive arterial thrombus formation in response to vascular injury that were highly unstable and prone to embolization in AC6 knockout mice.

Conclusion: These data demonstrate that AC6 is linked directly to PGI₂-mediated platelet inhibition and regulation of hemostasis and thrombosis in vivo.

Keywords: adenylyl cyclase; cAMP; hemostasis; platelets; thrombosis.