LPS and Platelet Activation in Myocardial Infarction
Condition: Myocardial Infarction; Acute Coronary Syndrome
MeSH terms: Myocardial Infarction; Acute Coronary Syndrome; Syndrome; Infarction
Study type: Observational

Summary: 
      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: 
      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.

      .
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        1. STEMI patients referred to the catheterization laboratory for PPCI, who will undergo
             to manual coronary thrombo-aspiration, that fulfilled the inclusion/exclusion
             criteria, with a sufficient thrombotic material (≥1 mm3).

          2. patients with chronic stable angina (SA) undergoing elective diagnostic and/or
             interventional coronary procedure, undergoing to intracoronary blood aspiration

          3. outpatients without coronary heart disease matched for age, gender and comorbidities
             like diabetes and hypertension
      
Biospecimen description: 
      Peripheral blood, aspirated intra-coronary blood, aspirated coronary thrombi, aspirated
      plaque fragments from coronary culprit lesions.
    
Keywords: Thrombi; LPS; Platelet Thrombus