Abstract 20618: Phosphorylation of Acetyl-coa Carboxylase by Ampk In Platelets Controls Thromboxane Generation, Dense Granules Secretion and Thrombus Formation

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Abstract

Introduction: AMPKα1 is activated in thrombin- or collagen-stimulated platelets. Acetyl-CoA carboxylase (ACC), the bona-fide substrate of AMPK, is phosphorylated/inhibited upon platelet stimulation but its role in platelets has never been investigated. ACC is a central regulator of lipid metabolism.

Hypothesis: Given the roles of lipids in platelets, namely structural, energy storage and signaling, we hypothesized that ACC phosphorylation could affect platelet functions and thrombus formation.

Methods: Platelets were isolated from a knock-in mouse model (KI) expressing a genetically modified ACC that can no longer be phosphorylated/inhibited by AMPK. In vitro, platelet adhesion and thrombus formation were measured using a flow chamber-based assay. Hemostasis was assessed via the measurement of bleeding time. Thrombosis was studied upon ferric chloride-induced carotid artery injury

Results: Platelets express the ACC1 isoform. Thrombin or collagen stimulation led to a rapid increase in ACC phosphorylation in WT platelets. However, baseline and thrombin- or collagen-induced ACC phosphorylation remained undetectable in KI platelets. In vitro, thrombus formation on collagen-coated surface was augmented under flow, in the absence of ACC phosphorylation. In vivo, KI mice had a shorter bleeding time and exhibited an increased thrombus growth reflected by a higher increment in thrombus surface area over a 10-minute time interval, compared to WT mice (KI,7242 μm2±1265; WT, 2520μm2±1219 for WT mice, P<0,05). Mechanistically, platelets from KI mice showed an increased collagen-induced thromboxane release and dense granules secretion (ATP release: KI=0.49 nmoles±0.03; WT=0.34 nmoles±0.05, P<0,05). Interestingly, lipidomic analysis revealed that KI platelets had increased levels of arachidonic-acid containing phosphatidylethanolamine plasmalogen, which are major contributors of arachidonic acid and thromboxane generation following platelet stimulation.

Conclusions: Platelet ACC phosphorylation by AMPK regulates phospholipids content which in turn, downregulates dense granules and thromboxane secretion, and thrombus formation. ACC phosphorylation is a counterregulatory mechanism limiting thrombus formation.

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