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Background Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Purpose Our study sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions by decreasing phospholipid content. Methods We generated a new Cre transgenic mouse strain that allows megakaryocyte/platelet specific ACC1 deletion (GpIbCre+/− x ACC1 flx/flx mouse). In vitro, platelet functions were assessed by aggregometry and flow cytometry. In vivo, hemostasis was assessed via the measurement of bleeding time. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Results As expected, ACC1 deletion was restricted to the megakaryocytic lineage. Hematological parameters in platelet-specific ACC1 knockout mice showed a decrease in platelet count by 30% and an increase in platelet volume by 31%, compared to ACC1 flx/flx platelets. In vitro, platelets from platelet-specific ACC1 knockout mice displayed a decrease in thrombin and CRP-induced platelet aggregation, associated with impaired dense granules secretion. In contrast, ADP-induced platelet aggregation was higher in the absence of ACC1. In vivo, platelet-specific ACC1 knockout mice showed a normal bleeding time. In agreement with our hypothesis, lipidomics analyses showed that ACC1 deletion in platelets was associated with a significant decrease in arachidonic acid-contaning phosphatidylethanolamine plasmalogen, and subsequently with a reduced production of thromboxane A2 upon thrombin or CRP stimulation. Conclusion Platelet-specific ACC1 deletion led to a decrease in phospholipid content which, in turn, decreased platelet thromboxane A2 generation, dense granules secretion and aggregation upon thrombin and CRP, but not ADP stimulation. Further studies are needed to elucidate the impact of ADP on platelet functions Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Summary: 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. Key Words: AMPK, acetyl-CoA carboxylase, coronary artery disease, lipidomics, platelet

Introduction Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. Purpose To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Methods Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Results Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production which could result from a decreased NOX2 activity. Conclusion Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Introduction Acetyl-CoA carboxylase (ACC) is the first committed enzyme regulating lipids synthesis. We have recently demonstrated that the overexpression of a constitutively active form of ACC in platelets leads to an increase in the phospholipid content which stimulates thrombus formation. Based on these data, we believe that ACC inhibition might be a counter-regulatory mechanism limiting endogenous lipogenesis and platelet reactivity in certain pathological circumstances. Aim Our study aims to assess the impact of pharmacological ACC1 inhibition on platelet functions, de novo lipogenesis and lipid content. Methods Platelets were treated with the long-chain fatty acid (FA) analog TOFA (30 μM) or the active site-directed CP640.186 (CP, 60 μM), two ACC inhibitors, for 2H before thrombin stimulation. Lipogenesis was measured via 14C-acetate incorporation into FA. Platelet functions were assessed by aggregometry. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Results Both ACC inhibitors decreased lipogenesis and thrombin-induced platelet aggregation. TOFA, but not CP, drastically decreased mitochondrial oxygen consumption rate, an effect associated with an increased reactive oxygen species production and AMPK activation. Surprisingly, the quantitative lipidomics analyses showed that a preincubation with CP did not affect global platelet lipid content. However, the short-term ACC inhibition (and potential acetyl-CoA accumulation) was sufficient to modify the level of acetylated proteins such as tubulin, a key player in the regulation of platelet shape change and aggregation. Conclusion Given the effect of TOFA on mitochondrial respiration, oxidative stress and AMPK activation, CP seems more appropriate to investigate the impact of ACC inhibition in platelets. A short-term acute treatment with CP inhibits thrombin-induced aggregation by a mechanism which doesn’t depend on lipid content but might involve changes in protein acetylation.

AMP-activated protein kinase (AMPK) α1 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.

Objectives Activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) leads to phosphorylation of acetyl-CoA carboxylase (ACC) on serine 79 (phosphoACC) and its subsequent inhibition. Given the role of AMPK-ACC signaling in platelet lipid metabolism and function, this pathway could be affected in the platelets of patients with coronary artery disease (CAD), where the atherogenic environment has an impact on platelet biology. We hypothesized that platelet AMPKACC signaling is activated by atherogenic lipids and could be considered as a metabolic signature in high-risk CAD patients. We also explored the consequences of platelet phosphoACC on platelet lipid metabolic profile. Approach and results Blood samples from 188 consecutive patients admitted for coronary angiography were processed. Lipid extracts from the platelets of 31 patients were subjected to lipidomic analysis. PhosphoACC level in circulating platelets of CAD patients were significantly increased and was linked to the severity of coronary artery calcification as well as acute coronary syndrome (OR: 6.71, 95% CI: 2.06–21.91; P = 0.002). The triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) ratio, a well-known atherogenic marker, was strongly associated with increased phosphoACC in our CAD cohort. Hence, oxLDL activated AMPK-ACC signaling through a CD36-dependent pathway. Lipidomic analysis revealed that increased phosphoACC led to a down-regulation of intraplatelet TG, particularly of those containing C14:0 fatty acid chains. Conclusion Platelet phosphoACC is a marker for risk stratification in suspected CAD patients and reveals an interaction between platelets and lipids. Inhibitory phosphoACC impacts platelet lipid content by down-regulating TG, which in turn may affect platelet function (ACCTHEROMA, NCT03034148).

Introduction Bioresorbable polymer stents have been used to provide a transient scaffold after coronary angioplasty. However, an increase in stent thrombosis has been observed. A current challenge is thus to develop new bioresorbable stents combining optimised mechanical and biodegradation properties together with limited thrombogenicity. Fe-based alloys are amongst the good candidates. Objective Blood compatibility of a new Fe-based alloy was studied in vitro via assessment of haemolysis and platelet activation. Methods Human whole blood was incubated for 60 min with either the Fe-based alloy or the cobalt-chromium (Co-Cr) alloy composing bare metal stents. After centrifugation, optical density of the supernatant was measured at 540 nm and the amount of haemolysis was calculated. For platelet activation assays, human or rat washed platelets were incubated for 60 min with both alloys before measuring their reactivity to platelet agonists by flow cytometry, using CD62P and activated α2bβ3 antibodies. In addition, phosphorylation of PKC substrates was evaluated by western blot. Results No significant red cells haemolysis was induced by both alloys (0.5 and 0.3% respectively). In addition, Co-Cr alloy did not affect CD62P exposure and α2bβ3 activation at platelet surface upon thrombin (0.03 to 0.3 U/ml) stimulation. In contrast, Fe alloy completely abolished their response to the agonist. A drastic inhibition of the phosphorylation state of PKC substrates was also observed after activation with thrombin, collagen (1.25 to 5 μg/ml) and ADP (0.1 to 10 μM). Since similar inhibitory effects were obtained when using a conditioned-reaction medium previously incubated with this Fe alloy, we postulate that its biocorrosion might release components counteracting platelet activation. Conclusion The Fe-based resorbable scaffold displays anti-thrombogenic properties and is a promising platform for next-generation stent technologies.

Introduction Bioresorbable polymer stents have been used to provide a transient scaffold after coronary angioplasty. However, an increase in stent thrombosis has been observed. A current challenge is thus to develop new bioresorbable stents combining optimized mechanical and biodegradation properties together with limited thrombogenicity. In this context Fe-based alloys are amongst the good candidates for stent manufacture. Objectives We aimed to explore in vitro blood compatibility of a new Fe-based alloy. Methods Human whole blood was incubated for 60 min with either the Fe-based alloy, pure iron, cobalt-chromium (CoCr) alloy (composing the bare metal stent used as a reference) or magnesium (Mg) alloy (composing a bioresorbable stent newly on the market). After centrifugation, haemolysis was calculated based on the optical density (540 nm) of the supernatant. For platelet activation assays, human washed platelets were incubated for 60 min with each metal before measuring their reactivity to a platelet agonist by flow cytometry, using CD62P and PAC-1 antibodies. In addition, phosphorylation of PKC substrates was evaluated by western blot. Results None of the alloys induced significant red cells haemolysis. In addition, Co-Cr did not affect CD62P exposure and PAC-1 presence at platelet surface upon thrombin (0.03 to 0.3 U/ml) stimulation. In contrast, Fe-based alloy and pure iron decreased significantly platelet response to agonist, as well as Mg but to a lesser extent. A drastic inhibition of the phosphorylation state of PKC substrates was also observed with the Fe-based alloy after activation with thrombin. Since similar inhibitory effects were obtained when using a conditioned-medium (incubated for 60 min with the alloy), we postulated that its bio-corrosion might release components counteracting platelet activation. Conclusion The Fe-based alloy seems to be a promising platform for next-generation stent technologies.

We compared the effects of total thyroidectomy (TTx) and radioiodine (RAI) administration on the course of thyroid hormones and thyroid-stimulating immunoglobulins (TSI) in patients with Graves' disease.We retrospectively studied 80 patients initially treated with antithyroid drugs and requiring either RAI (8.3 ± 1.7 mCi of (131)I; n = 40) or TTx (n = 40) as second-line therapy.The TTx and RAI groups were not different, except for larger goiter, higher FT3 and more frequent Graves' orbitopathy at diagnosis in the surgery group (p0.05). A persistent remission of hyperthyroidism was observed in 97% of operated patients versus 73% of the RAI patients at 3 years (p0.01). TTx was followed by a rapid and steady decrease in TSI during the first 9 months, while a surge of antibodies was observed during the first 6 months after RAI, followed by a slow decrease over the next 18 months. At the last visit, high TSI levels were still observed in 18 and 60% of patients in the surgery and RAI groups, respectively (p0.001).TTx is more efficient than RAI to induce a rapid and permanent correction of hyperthyroidism and TSI decrease in patients previously treated with antithyroid drugs.

A 53-year-old male was admitted for typical acute chest pain. The ECG showed a mirror image of posterior myocardial ischemia. Initial biology was normal but cardiac markers (creatine kinase and troponin) rose later. Echocardiography did not reveal any hypokinetic myocardial segment. There was no left ventricular dysfunction or valvular disease. There was no pericardial effusion or aortic dissection image. This patient was treated as a “non-ST segment elevation myocardial infarction” (NSTEMI), also called subendocardial myocardial infarction. A selective coronary angiography (SCA) was performed the next day and after careful examination by several experts, no coronary lesion was detected. Left ventriculography was also normal. Cardiac MRI was then performed and revealed a late focal subendocardial enhancement, located in the mid infero-posterior myocardial segment (Fig. A, arrow). This lesion appeared to be ischemic, despite normal SCA. Computed tomography coronary angiography (CTCA) was finally done, showing a hypodense image, with also an ischemic aspect, in the same subendocardial area (Fig. B, arrow) as observed on MRI. Furthermore, CTCA detected tight luminal narrowing with hypodense material (soft atheroma or clot) in a circumflex branch (Fig. C, arrow), corresponding to the suspected ischemic territory. In this case, CTCA both confirmed ischemic etiology and identified culprit artery missed by SCA.