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PLatelets Acetyl-CoA Carboxylase Phosphorylation State in SEPtic Shock

2019-10-03 07:54:48 | BioPortfolio

Summary

Knowing the dramatic increase in thrombin generation during sepsis, our research hypothesis is that AMPK-induced ACC phosphorylation in platelets is increased and that this might modulate platelets metabolism and more particularly platelets inflammatory mediators content, coming from AA and lipids.

Description

Background

Sepsis, a systemic inflammatory reaction occurring in response to an infection, is a major public health problem and is recognized as the leading cause of death in the intensive care unit (ICU). This severe inflammatory reaction causes endothelial activation and dysfunction. This phenomenon is closely associated with the activation of coagulation cascade via exposure of tissue factor (TF) at endothelial cell surface. TF serves as an anchor for factors of coagulation VII and X allowing their activation. This results in thrombin (IIa) generation and fibrin production.

Thrombin is also a potent platelet agonist (activator) resulting in platelet activation and consumptionThe pro-coagulant activity of activated platelets during sepsis enhances the formation of micro-thrombosis that impair organ perfusion and may lead to death. Thrombocytopenia is common and is a strong negative prognostic marker in septic shock patients.

Beyond coagulation, platelets are known to play a key role in inflammation and immune response. Platelets activation can be initiated by inflamed endothelium or circulatory inflammatory cytokines, which in turn, modulates the inflammatory and thrombotic response. Activated platelets interact with immune cells, like neutrophils. Furthermore, platelets can act as microbial "sensors" by expressing members of Toll-Like Receptors (TLRs) family, binding ligands from several infectious agents. Recent data highlight the interplay between platelets and neutrophils extracellular traps (NETs). Platelets facilitate NETs formation and, inversely, NETS activate platelets. NETs connect platelets, thrombosis, immune response and inflammation and therefore are of particular interest in the septic context.

Platelets express Acetyl-CoA carboxylase 1 (ACC1), which is the first step enzyme of lipogenesis. ACC Phosphorylation on Serine 79 by AMP-activated protein kinase (AMPK) leads to its inhibition and AMPK is activated by thrombin. We demonstrated that AMPK-ACC axis controls platelets phospholipids content, which influence TXA2 and dense granule release and, in turn, thrombus formation. TXA2 is generated from phospholipids containing arachidonic acid (AA). Alternatively, AA can be metabolized by lipooxygenase (LOX) pathway producing lipoxins (LX) and resolvin, which are rather involved in the resolution of inflammation. The relation between AMPK-ACC signalling and lipooxygenase pathway has never been investigated.

Research hypothesis

Knowing the dramatic increase in thrombin generation during sepsis, our research hypothesis is that AMPK-induced ACC phosphorylation in platelets is increased and that this might modulate platelets metabolism and more particularly platelets inflammatory mediators content, coming from AA and lipids.

Patients selection

Consecutive patients with septic shock admitted at Cliniques universitaires Saint-Luc, Brussels, will be included (experimental group)

Control group will correspond to healthy volunteers with matched age and gender, based on retrospective analysis of patients admitted in ICU for sepsis.

Exploratory control group will correspond to patients admitted in the intensive care unit without evidence of severe infection and severe systemic inflammatory response. Patients with neurological disorder and intoxication will be the target population.

Similar exclusion criteria will be the same in control groups than in the experimental group.

Material and methods

Type of study Prospective, monocentric, interventional study. Blood sampling Blood samples will be collected after venous catheter insertion, within 48 hours after septic shock diagnosis.

The following samples will be taken during the procedure:

- Coagulation including (INR, TCA, TT, PTT, Fibrinogen, DDimers) (1 green TUBE of 3 ml)

- Multiplate analysis (1 orange TUBE of 3 ml)

- Blood sample for platelets protein analysis (including ACC phosphorylation). After platelets isolation, the remaining plasma will be frozen (2 TUBES CPDA of 8.5 ml).

- Urine sample. Data collection Data will be collected from Medical Explorer and Q Care, including biological data that are routinely performed in patients admitted in the ICU as platelets count, CRP level, coagulation assessment, renal function and liver enzymology. Follow-up will be performed by at least 3 years and no additional visits will be planned. Events recorded during the follow-up period will be obtained from Medical Explorer and a phone call will be done in case of missing data.

Measurements

- Blood cells count including platelets.

- ACC phosphorylation via western blotting, ECLIA.

- Platelets protein extract for protein acetylation.

- Platelet function ex vivo using Multiplate analysis.

- Plasma sampling.

- Platelets lipidomics analysis. ACC phosphorylation analysis

- Plasma-rich-platelet (PRP) will be obtained after centrifugation. Apyrase and Integrilin are added to limit platelet activation during preparation. PRP will be divided into 3 samples and platelets will be pelleted after centrifugation at 400 g for 10 minutes.

- One of the 3 platelets samples will be lysed with Lemli solution for Western blot analysis of phosphorylated ACC, ACC1 and phosphorylated protein kinase C substrates. All samples will then be stocked at -80°c. In order to compare different immunoblotting, control platelets samples are obtained after thrombin stimulation. Each patient's platelet sample will be compared to the same control sample. The signal of phosphorylated ACC for each patient will be quantified by Image J (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014) and will be expressed as a fraction of the control sample signal.

- Level of phosphorylated ACC will be confirmed by electrochemiluminescence (ECLIA, Meso Scale Discovery) and flow cytometry (FACS).

- Protein extracts will be used for platelets protein analysis using immunoblotting.

Plasma sampling

- Coagulation markers including INR, TCA, TT, PTT, Fibrinogen, D-Dimers, Thrombin antithrombin complex (TAT).

- Cytokines measurements (TNF-alpha, IL-1b, IL-6, chemokines- CCL 3, 5, and 18, complement system) as well as inflammatory biomarkers (hypersensitive C-reactive protein).

- Platelets activation biomarkers (sCD62P, sCD40L, CD62P, PF4) (collaboration with Dr C. Oury, GIGA, Université de Liège, Belgium).

- Fibrinolytic biomarkers (u-PA, t-PA and PAI).

- Lipidomic. Urines Samples of urines will be collected in the same times that blood sampling to measure TX2B generation.

Lipidomic analyses Lipidome will be analyzed in collaboration with Christine Des Rosiers in Montréal Heart Insitute. Special attention will be given to metabolites from the COX and LOX pathways (TXA2 and lipoxins).

Sample size

Based on our preliminary data, we determined that enrolment of minimum 46 patients would provide a power of 90% at a significance level of 5% to detect a difference of 0.15 in the phosphorylation of ACC (difference between control group 1 and septic shock population). 20 patients are expected in the exploratory control group.

Study Design

Conditions

Septic Shock

Intervention

Platelets Acetyl-CoA Carboxylase phosphorylation analysis

Location

Cliniques Universitaires St Luc
Bruxelles
Belgium
1200

Status

Recruiting

Source

Cliniques universitaires Saint-Luc- Université Catholique de Louvain

Results (where available)

View Results

Links

Published on BioPortfolio: 2019-10-03T07:54:48-0400

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Medical and Biotech [MESH] Definitions

A carboxylating enzyme that catalyzes the conversion of ATP, acetyl-CoA, and HCO3- to ADP, orthophosphate, and malonyl-CoA. It is a biotinyl-protein that also catalyzes transcarboxylation. The plant enzyme also carboxylates propanoyl-CoA and butanoyl-CoA (From Enzyme Nomenclature, 1992) EC 6.4.1.2.

The late onset form of MULTIPLE CARBOXYLASE DEFICIENCY (deficiency of the activities of biotin-dependent enzymes propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, and PYRUVATE CARBOXYLASE) due to a defect or deficiency in biotinidase which is essential for recycling BIOTIN.

A deficiency in the activities of biotin-dependent enzymes (propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, and PYRUVATE CARBOXYLASE) due to one of two defects in BIOTIN metabolism. The neonatal form is due to HOLOCARBOXYLASE SYNTHETASE DEFICIENCY. The late-onset form is due to BIOTINIDASE DEFICIENCY.

The neonatal form of MULTIPLE CARBOXYLASE DEFICIENCY that is caused by a defect or deficiency in holocarboxylase synthetase. HLCS is the enzyme that covalently links biotin to the biotin dependent carboxylases (propionyl-CoA-carboxylase, pyruvate carboxylase, and beta-methylcrotonyl-CoA carboxylase).

A subfamily of small heat-shock proteins that are closely related to ALPHA B-CRYSTALLIN. Hsp20 heat-shock proteins can undergo PHOSPHORYLATION by CYCLIC GMP-DEPENDENT PROTEIN KINASES.

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