RT Journal Article SR Electronic T1 Acetylcholine inhibits platelet activation and regulates hemostasis JF bioRxiv FD Cold Spring Harbor Laboratory SP 324319 DO 10.1101/324319 A1 John A. Bennett A1 Sara K. Ture A1 Rachel A. Schmidt A1 Michael A. Mastrangelo A1 Scott J. Cameron A1 Lara E. Terry A1 David I. Yule A1 Craig N. Morrell A1 Charles J. Lowenstein YR 2018 UL http://biorxiv.org/content/early/2018/05/16/324319.abstract AB Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but there are fewer identified endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO). Acetylcholinesterase inhibitors such as donepezil can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets.We measured the effect of acetylcholine or analogues of acetylcholine upon human platelet activation ex vivo. We characterized expression of components of the acetylcholine signaling pathway in human platelets. We tested the effect of a subunit of the acetylcholine receptor, CHRNA7, on acetylcholine signaling in platelets. Acetylcholine and analogues of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and GPIIbIIIA conformational changes. Conversely, we found that antagonists of the acetylcholine receptor such as pancuronium enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase such as donepezil also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Human platelets express members of the acetylcholine signaling pathway including CHRNA2, CHRNA7, CHRNB1, and ACHE. Platelets from mice lacking Chrna7 are hyperactive when stimulated by thrombin and resistant to inhibition by acetylcholine. Furthermore, acetylcholinesterase inhibitors prolonged bleeding in wild-type mice. Knockout mice lacking Chrna7 subunits of the acetylcholine receptor display prolonged bleeding as well.Our data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for anti-thrombotic therapies.