LPS and Platelet Activation in Myocardial Infarction

Study Overview
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Brief Summary
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Platelets play a key role in the athero-thrombotic process. However, the in vivo mechanism accounting for thrombus growth at site of coronary atherosclerotic lesion has not been fully elucidated. While platelet adhesion and aggregation on the thrombogenic core of atherosclerotic plaque is an established mechanism for thrombus growth, the role of systemic factors, which may contribute to thrombus via amplification and propagation of platelet aggregation, is still to be clarified. There is a growing body of evidence that lipopolysaccharides (LPS), are implicated in athero-thrombosis. Circulating levels of endotoxins have been associated with human atherosclerosis progression, particularly in smokers or in patients with infections. Furthermore, endotoxins seem to be implicated in the thrombotic process through several mechanisms including up-regulation of macrophage tissue factor expression and amplification of platelet response upon interaction with Toll-like receptor 4. The relationship between endotoxins and platelets may be relevant in the context of acute coronary syndromes as endotoxins could locally amplify platelet-derived thrombus growth but this issue is still unexplored. Previous studies demonstrated that low-grade endotoxemia is detectable in human circulation, likely as consequence of enhanced gut permeability, and may be responsible for leucocyte-platelet aggregate and eventually thrombosis. The investigators hypothesize that low-grade endotoxemia may be observed in patients with coronary heart disease and may favor, at site of coronary unstable plaque, thrombus growth. To explore this issue, Escherichia Coli (EC)-LPS concentration and biomarkers of platelet activation will be measured in coronary thrombus and intra-coronary blood of patients with STEMI and stable angina (SA), respectively, and in peripheral circulation of both patients and controls. EC DNA will be searched in serum of all patients by polymerase chain reaction (PCR). Furthermore, to substantiate that LPS could be biologically active, immune-histochemical analysis of thrombi and in vitro studies will be performed to assess the interplay between LPS and platelet activation.

Detailed Description
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In this case-control study, three groups of patients will be compared: consecutive STEMI patients undergoing to manual thrombo-aspiration during primary percutaneous coronary intervention, patients with chronic stable angina (SA) undergoing elective diagnostic and/or interventional coronary procedure and outpatients without coronary heart disease referring to the ambulatory of the Department of Internal Medicine, I Clinica Medica, Sapienza -University of Rome. Patients will be recruited from three Centers: i) Department of the Heart and Great Vessels Attilio Reale, Sapienza -University of Rome; ii) Department of Internal Medicine, I Clinica Medica, Sapienza -University of Rome; iii) Department of Interventional Cardiology, Santa Maria University Hospital, Terni. The study complied with the Declaration of Helsinki and was approved by the local ethic committees of centers involved. In patients presenting STEMI, coronary thrombi, when present, or plaque fragments will be aspirated from the culprit coronary artery before stent implantation and collected in EDTA tubes. Thrombi will be homogenized in 5 mL of a homogenization buffer. Aliquots of thrombi homogenate will be centrifuged. In a subset of STEMI patients, part of the thrombotic material aspirated will be fixed in 4% buffered formaldehyde for histologic and immunohistochemical analyses. In patients with SA, intracoronary blood will be aspirated from the stented coronary artery, before stenting, and immediately collected in EDTA tubes and centrifuged. Next, supernatant will be removed and stored at -80°C until use. Peripheral blood samples will be obtained from a radial or femoral artery, before the start of procedure and after stent deployment in STEMI patients, or before balloon dilation and stenting in SA patients and then collected into tubes with or without 3.8% sodium citrate and EDTA tubes and centrifuged to obtain supernatant. Blood samples of controls group will be obtained from patients after supine rest for at least 10 min and taken into tubes with or without 3.8% sodium citrate and in EDTA tubes and centrifuged to obtain supernatant. Plasma and serum aliquots will be stored at -80°C in appropriate cuvettes until assayed. Complete haemochrome, blood glucose, lipid profile, fibrinogen, creatinine, creatine kinase-MB and troponin T will be evaluated using standard methods. sCD40L and sP-selectin levels will be measured with a commercial immunoassay in aliquots of plasma, thrombus homogenate and intracoronary blood. Lipopolysaccharide (LPS) levels in serum and thrombus will be measured using a commercial ELISA kit. A PCR reaction for specific amplification of a region of the 16S ribosomal RNA gene of Escherichia coli will be developed. Serum zonulin levels will be measured using a commercial ELISA kit. Immunoistochemistry (IHC) will be performed on sections obtained from formalin-fixed and paraffin embedded thrombus fragments aspirated from a subset of STEMI patients. After rehydration and antigen retrieval slides will be incubated with primary antibodies respectively to LPS, TLR4 and Cathepsin G, then washed in phosphate saline buffer and incubated with a secondary universal antibody. Immunoreactions will be detected with diaminobenzidine. .

Official Title
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Endotoxemia in Coronary Thrombus of Patients With Acute Coronary Syndrome

Conditions
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Myocardial Infarction, Acute Coronary Syndrome

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: For STEMI patients: diagnosis of STEMI based on the current European Guidelines For SA patients: diagnosis of SA defined according to the European Guidelines as lack of episodes of coronary instability for at least 6 months prior to admission For control subjects: outpatients without diagnosis of coronary heart disease Exclusion Criteria: estimated glomerular filtration rate less than 30 ml/min/m2 acute or recent systemic infections (3 weeks) treatment with systemic corticosteroids treatment with oral anticoagulants malignancy lack of consent to participate Additional exclusion criteria for STEMI patients were symptoms duration>12 h, rescue PCI, in-stent thrombosis and anatomical difficulty in reaching the lesion.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 95 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| STEMI<br>50 STEMI patients treated with standard therapy undergoing to primary percutaneous coronary internention (PPCI). Thromboaspiration will be performed whenever possible (when the anatomy of the coronary artery - curve and size- allowed it) in all patients with a TIMI Flow 0 and in all patients with a visible thrombus if TIMI Flow was 1 or more. | |
| Stable angina<br>50 stable angina (SA) patients on standard therapy, undergoing to intracoronary blood aspiration during elective diagnostic and/or interventional coronary procedure, matched for age, sex and comorbidities with the 50 STEMI patients. | |
| Controls<br>50 outpatients without coronary heart disease, matched for age gender and comorbidities like diabetes and hypertension with the 50 STEMI patients. Peripheral blood samples will be collected during routine patient monitoring. | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| LPS in blood of STEMI, SA patients and controls. | LPS will be measured in serum and expressed as concentration (pg/ml) | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| LPS in thrombus and intra-coronary blood of STEMI and SA patients. | LPS will be measured in coronary thrombi of STEMI patients and in intra-coronary blood of SA patients and expressed as concentration (pg/ml) | 1 year | 
| sP-selectin in thrombus and intra-coronary blood of STEMI and SA patients. | sP-selectin (a marker of platelet activation) will be measured in coronary thrombi of STEMI patients and in intra-coronary blood of SA patients and expressed as concentration (ng/ml). | 1 year | 
| sCD40L in thrombus and intra-coronary blood of STEMI and SA patients. | sCD40L (a marker of platelet activation) will be measured in coronary thrombi of STEMI patients and in intra-coronary blood of SA patients and expressed as concentration (ng/ml). | 1 year | 
| Escherichia coli-DNA | Escherichia coli-DNA will be searched in serum patients and controls by polymerase chain reaction (PCR).The investigators will evaluate the rate of positivity in the serum of the study population. | 1 year | 
| Histologic and immunohistochemical analyses of thrombus fragments aspirated from a subset of STEMI patients. | Immunoistochemistry on sections obtained from formalin-fixed and paraffin embedded thrombus fragments.The investigators will analyze the composition of thrombus fragments and the presence of LPS , TLR4 and Cathepsin G. | 1 year | 
| HS-CRP in blood of STEMI, SA patients and controls | High sensitivity-C reactive protein will be measuer in serum and expressed as concentration (mg/L). | 1 year | 
| sP-selectin in blood of STEMI, SA patients and controls. | sP-selectin (a marker of platelet activation) will be measured in plasma and expressed as concentration (ng/ml) | 1 year | 
| Soluble CD40L (sCD40L) in blood of STEMI, SA patients and controls. | sCD40L (a marker of platelet activation) will be measured in plasma and expressed as concentration (ng/ml) | 1 year | 
| Zonulin in blood of STEMI, SA patients and controls | Zonulin (a marker of gut permeability) will be measued in serum and expressed as concentration (ng/ml) | 1 year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Thrombi, LPS, Platelet Thrombus