Exploring the role of AMPK-ACC signalling and lipid metabolism in platelet functions and thrombus formation

Platelets are central actors in haemostasis and thrombosis. Their stimulation by thrombin or collagen induces the phosphorylation and inhibition of the acetyl-CoA carboxylase (ACC). Since ACC is crucial for the synthesis of fatty acids, which are essential for platelet structure, signalling and metabolism, we hypothesized that this enzyme plays a role in platelet function. We showed that constitutive ACC activation increases thromboxane generation, leading to enhanced platelet reactivity and thrombus formation. Lipidomic analysis of intraplatelet content revealed an increase in arachidonic acid-containing phospholipids, which are major precursors of thromboxane. Interestingly, ACC is phosphorylated in high-risk coronary artery disease patients.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Bioreactivity of Stent Material: In Vitro Impact of New Twinning-Induced Plasticity Steel on Platelet Activation

A current challenge concerns developing new bioresorbable stents that combine optimal mechanical properties and biodegradation rates with limited thrombogenicity. In this context, twinning-induced plasticity (TWIP) steels are good material candidates. In this work, the hemocompatibility of a new TWIP steel was studied in vitro via hemolysis and platelet activation assessments. Cobalt chromium (CoCr) L605 alloy, pure iron (Fe), and magnesium (Mg) WE43 alloy were similarly studied for comparison. No hemolysis was induced by TWIP steel, pure Fe, or L605 alloy. Moreover, L605 alloy did not affect CD62P exposure, αIIbβ3 activation at the platelet surface, or phosphorylation of protein kinase C (PKC) substrates upon thrombin stimulation. In contrast, TWIP steel and pure Fe significantly decreased platelet response to the agonist. Given that similar inhibitory effects were obtained when using a conditioned medium previously incubated with TWIP steel, we postulated TWIP steel corrosion to be likely to release components counteracting platelet activation. We showed that the main ion form present in the conditioned medium is Fe3+. In conclusion, TWIP steel resorbable scaffold displays anti-thrombogenic properties in vitro, which suggests that it could be a promising platform for next-generation stent technologies

0382: AMPKalpha1 regulates actin polymerization, lamellipodia formation and clot retraction, in thrombin-stimulated platelets

BackgroundPlatelet activation requires sweeping morphological changes, supported by contraction and remodelling of platelet actin cytoskeleton. In epithelial and endothelial cells, AMP-activated protein kinase (AMPK) controls actin cytoskeleton organization through the phorphorylation of cytoskeletal targets, namely myosin regulatory light chains (MLC), cofilin and the vasodilator-stimulated phosphoprotein (VASP), extending the role of AMPK beyond metabolism.ObjectivesIn this study, we hypothesized that AMPK was activated in thrombin-stimulated platelets and played a role in platelet secretion, aggregation and clot retraction, by regulating polymerization and/or organization of actin cytoskeleton through the phosphorylation of MLC, cofilin and VASP.ResultsHuman platelets expressed exclusively the AMPKalpha1 isoform. In human purified platelets, thrombin led to a transient activation of AMPKalpha1 and to phosphorylation of its bona fide substrate, acetyl coA carboxylase (ACC). Platelets isolated from mice lacking AMPKalpha1 exhibited reduced aggregation and secretion response to thrombin, associated with a defect in ACC, MLC, cofilin and VASP phosphorylation. These changes were associated with an abrogration of thrombin-dependent F-actin formation. Moreover, the percentage of platelets able to form lamellipodia after immobilization on fibrinogen-coated coverslips and stimulation by thrombin, was significantly reduced in the absence of AMPKalpha1, indicating an altered cytoskeleton reorganization during spreading. More importantly, clot retraction was slower and less effective in KO platelets.ConclusionsAMPKalpha1 plays a critical role in platelet function in response to thrombin through the phrophorylation of cytoskeletal targets and the subsequent regulation of cytoskeleton organization-dependent processes

Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation.

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton

Hypothermic continuous machine perfusion enables preservation of energy charge and functional recovery of heart grafts in an ex vivo model of donation following circulatory death.

OBJECTIVES: Cardiac transplantation using hearts from donors after circulatory death (DCD) is critically limited by the unavoidable warm ischaemia and its related unpredictable graft function. Inasmuch as hypothermic machine perfusion (MP) has been shown to improve heart preservation, we hypothesized that MP could enable the use of DCD hearts for transplantation. METHODS: We recovered 16 pig hearts following anoxia-induced cardiac arrest and cardioplegia. Grafts were randomly assigned to two different groups of 4-h preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems©, Itasca, Il). After preservation, the grafts were reperfused ex vivo using the Langendorff method for 60 min. Energetic charge was quantified at baseline, post-preservation and post-reperfusion by measuring lactate and high-energy phosphate levels. Left ventricular contractility parameters were assessed both in vivo prior to ischaemia and ex vivo during reperfusion. RESULTS: Following preservation, the hearts that were preserved using CS exhibited higher lactate levels (57.1 ± 23.7 vs 21.4 ± 12.2 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate ratio (0.53 ± 0.25 vs 0.11 ± 0.11; P < 0.001) and lower phosphocreatine/creatine ratio (9.7 ± 5.3 vs 25.2 ± 11; P < 0.001) in comparison with the MP hearts. Coronary flow was similar in both groups during reperfusion (107 ± 9 vs 125 ± 9 ml/100 g/min heart; P = ns). Contractility decreased in the CS group, yet remained well preserved in the MP group. CONCLUSION: MP preservation of DCD hearts results in improved preservation of the energy and improved functional recovery of heart grafts compared with CS

HYPOTHERMIC CONTINUOUS MACHINE PERFUSION IMPROVES METABOLIC PRESERVATION AND FUNCTIONAL RECOVERY IN DCD HEART GRAFTS.

Background: the number of heart transplants is declining since the 2000s because of organ shortage. Donation after cardiac death (DCD) is an alternative source of grafts. Hearts from DCD donors are not transplanted due to warm ischemia and possible poor graft function. Hypothermic machine perfusion (MP) has proven to be superior to cold storage (CS) for the preservation of kidneys, livers or lungs from DCD. We investigated in a large animal mod-el whether MP improves preservation of DCD hearts and allow its use for transplantation. Method: 16 pig hearts were retrieved after anoxia induced cardiac arrest and cardioplegia. They were randomized in 2 groups: 8 hearts preserved on CS; 8 hearts on MP (Modified Lifeport© System, Organ Recovery Systems©, Itasca, Il). The preservation solution was KPS-1 in both groups. After a period of 4 hours, the grafts were reper-fused on a Langendorff during 60 min. Myocardial biopsies were performed at baseline, post-preservation and post-reperfusion to quantify oxidative metabolism by measuring lactate, nucleotides, phosphocreatine (Pcr) and creatine (Cr). Contractility index (CI) was assessed in vivo before cardioplegia with a Millar catheter and during reperfusion with an intraventricular balloon. Results: compared to MP, hearts preserved on CS showed significant higher lactate levels (57,1±23,4 vs 21,4±12,2 μmoles/g heart; p<0,001), higher AMP/ATP ratio (0,53±0,25 vs 0,11±0,11; p<0,0001) and lower PCr/Cr ratio (9,7±5,3 vs 25,2±11; p<0,001) after preservation. Compared to baseline, CI decreased by 26±7% the CS group while it remained unchanged in the MP group (p<0,05). Conclusion: Compared to MP, DCD hearts preserved on CS suffer more severe ischemia during preservation, as shown by a higher lactate and lower energy states. Grafts preserved on MP recovered well after reperfusion, while function was depressed in grafts preserved on CS

Heart grafts from DCD display acceptable metabolic preservation and functional recovery compared to BDD.

Background: the number of heart transplants is declining since the 2000s because of organ shortage. Donation after cardiac death (DCD) is an alternative source of grafts, but hearts from DCD are not currently utilized due to concerns that they may suffer irreversible cardiac injury with subsequent poor post-transplant function. However, little is known about the quality of those grafts. In this large animal model we compared metabolic preservation and functional recovery of grafts from DCD compared with grafts from brain death donors (BDD). Method: 16 pig hearts were randomized in two groups. In the BDD group, hearts were classically retrieved after cardioplegia. In the DCD group, hearts were recovered after anoxia induced cardiac arrest and cardioplegia. All were preserved on ice for a period of 4 hours. The preservation solution was KPS-1 in both groups. After preserva-tion, the grafts were reperfused on a Langendorff during 60 min. Myocardial biopsies were performed at baseline, post-preservation and post-reperfusion to quantify oxidative metabolism by measuring lactate, nucleotides and phosphocreatine (Pcr). Contractility index (CI) was assessed in vivo before cardioplegia with a Millar catheter and during reperfusion with an intraventricular balloon. Results: Warm ischemic time (WIT) was 0 min in BDD vs 14±2 minutes in DCD (p<0,001). Compared to grafts from BDD, hearts from DCD showed significant lower lactate levels (54,6±23,6 vs 23,7±24,7 μmoles/g heart; p<0,001), similar ATP concentration (8,4±3,1 vs 7,6±2,8 μmoles/g heart; p=ns) and similar PCr concentration (8,0±2,5 vs 9,2±4,6; p=ns) after preservation. Compared to baseline, CI decreased by 25±7 in the BDD group and 26±7% in the DCD group (p=ns). Conclusion: In this ex vivo model, DCD hearts showed similar preservation of the energy states and contractility after reperfusion compared to hearts from BDD, despite the warm ischemic period. This could be explained by is-chemic preconditioning during WIT

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) alpha1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation

Platelet Acetyl-CoA Carboxylase Phosphorylation A Risk Stratification Marker That Reveals Platelet-Lipid Interplay in Coronary Artery Disease Patients

Adenosine monophosphate–activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC) signaling is activated in platelets by atherogenic lipids, particularly by oxidized low-density lipoproteins, through a CD36-dependent pathway. More interestingly, increased platelet AMPK–induced ACC phosphorylation is associated with the severity of coronary artery calcification as well as acute coronary events in coronary artery disease patients. Therefore, AMPK–induced ACC phosphorylation is a potential marker for risk stratification in suspected coronary artery disease patients. The inhibition of ACC resulting from its phosphorylation impacts platelet lipid content by down-regulating triglycerides, which in turn may affect platelet function