Platelet Acetyl Coenzyme A Carboxylase Phosphorylation in Coronary Artery Disease
Platelet Acetyl Coenzyme A Carboxylase Phosphorylation in Coronary Artery Disease
In human purified platelets, only thrombin, and not the other platelet agonists, leads to a
 transient activation of the protein kinase activated by AMP (AMPK) and to phosphorylation of
 its "bona fide" substrate, ACC on its Ser79. ACC phosphorylation (P-ACC) can be an
 interesting marker of thrombin action on platelets. Indeed platelet and coagulation
 interplay, though undoubtedly present in atherosclerosis and atherothrombosis, remains
 difficult to assess. Our group showed that atherosclerotic mice (SRBI/Apolipoprotein E
 knock-out) had higher platelet P-ACC compared to corresponding control mice (C57BL6). In
 agreement with these data, preliminary results showed increased platelet P-ACC in a small
 cohort of patients admitted for coronary angiogram, with demonstrated coronary artery disease
 (CAD).

 In the light of our preliminary results, we sought to analyze platelet P-ACC in a large
 prospective clinical trial (ACCTHEROMA) in patients admitted for coronary angiogram. The aim
 of the study is to compare platelet P-ACC in platelets of patients with CAD and more
 particularly in unstable CAD patients to non-CAD patients. This study could potentially
 identify patients at high risk of future ischemic cardiovascular events, because of a higher
 level of thrombin generation.
Consecutive patients with planned coronary angiogram at the Cliniques Universitaires Saint
 Luc, Brussels, whatever the indication, will be included between March 2015 and February
 2016. Informed consent will be obtained before angiogram.

 A. Blood Sampling Routine veinous blood sample will be done under fasting condition in the
 cardiology ward before the angiogram (standard of care for patients undergoing coronary
 angiogram).

 In order to avoid additional puncture for blood sampling and to limit platelet activation,
 blood samples are withdrawn after sheath insertion at the Cath lab before any drug
 administration. The following measurements will be done:

 -  Routine coagulation tests including international rationalize ratio, activated cephaline
 time, thrombin time, prothrombin time, Fibrinogen, DDimers.

 -  Platelet reactivity testing by the Multiple Electrode Aggregometer (Multiplate Analysis)

 -  Platelet isolation and lysates will be stocked frozen for phospho-protein analysis by
 immunoblotting and electrochemiluminescence (ECLIA)

 -  Aliquots of poor platelet plasma stocked frozen for inflammation (HsCRP), thrombin
 generation tests (thrombin-antithrombin complex, prothrombin fragment F1.2, endogenous
 thrombin generation potential-ETP), platelet activation markers (platelet factor 4,
 soluble CD40ligand, soluble P-selectin) and circulating micro-RNA extraction.

 -  Platelet isolation and further purification for platelet specific micro-RNA extraction.

 B. Atherosclerosis evaluation All patients underwent coronary angiogram. Significant coronary
 artery lesion is defined as a luminal stenosis above 50%. Syntax score will be calculated for
 all patients.

 Global atherosclerotic plaque burden on coronary and extracoronary arteries will be evaluated
 by measuring calcium score on thoraco-abdominal scanner with prospective ECG-gating in a
 randomly selected subgroup of patients. Coronary artery calcification (CAC) is expressed as
 Agatston score using calcium scoring software (Philips Healthcare) with a threshold of 130
 Hounsfield units. The degree of atherosclerotic burden on coronary arteries is classified as
 mild (Agatston score <100), moderate (Agatston score 100-400) or severe (Agatston score
 >400). Aortic calcification (AoC score) was measured from aortic arch to iliac arteries in
 all patients.

 C. Data collection Baseline characteristics of patients will be collected from the medical
 database of the Cliniques Universitaires Saint Luc. All coronary angiogram will be reviewed
 by 2 different investigators and syntax score will be calculated with the online syntax score
 calculator. CAC Agatston and AoC score will also be assessed by 2 different investigators.

 D.Patients Classification for platelet P-ACC analysis Platelet ACC phosphorylation (P-ACC)
 analysis will be done by immunoblotting with a specific antibody probing for phosphorylated
 (Ser 79) ACC. Purified washed platelets stimulated with 0.5 units/ml thrombin will be used as
 positive internal controls.

 Patients will first be classified in non-CAD and CAD groups based on the results of coronary
 angiogram and CAC Agatston score: Non-CAD (absence of lesion on angiogram and/or CAC Agatston
 score<100) and CAD (presence of at least 1 lesion on angiogram and/or CAC Agatston
 score>100).

 In a second analysis, based on clinical presentation, the CAD group will further be divided
 into Stable CAD or Unstable CAD [acute coronary syndrome (ACS)]. Platelet P-ACC will be
 compared in both subgroups. This detailed classification will outline high-risk patients with
 ongoing acute coronary syndrome.

 E. Sample size and statistical analysis. Based on our preliminary data, we determined that
 enrolment of 102 patients would provide a power of 80% at a significance level of 5% to
 detect a difference of 0.15 (A.U.) in the phosphorylation of ACC in atherosclerotic patients,
 compared to non CAD patients.

 Continuous variables will be expressed as mean +/- one standard deviation, categoric
 variables as counts and percentages. Patients will separated in four groups depending on the
 level of P-ACC (quartiles). Clinical presentation, thrombin generation markers and calcium
 scoring will be compared in all these groups, based on ANOVA analysis. Variables will be
 submitted to logistic regression test in order to identify independent predictors of high ACC
 phosphorylation state. The role of high platelets P-ACC in assessing the risk of the patients
 will be then studied.

 F.Follow-up A follow-up period of 3 years is planned. Events recorded during the follow-up
 period will be obtained from the medical database and a phone call will be done in case of
 missing data.
Inclusion Criteria:

 -  Signed informed consent

 -  Planned angiography within the next 3 days, whatever the indication.

 Exclusion Criteria:

 -  Not able to sign informed consent

 -  Patients on anticoagulation therapy (oral or parenteral) including heparins,
 fondaparinux, vitamin K antagonist, novel oral anticoagulants, for any reasons.

 -  Active neoplasia

 -  Active inflammatory disease

 -  Patients with life expectancy lower than 3 years

 -  Haemophilia and other coagulopathy

 -  Abnormal platelet count

 -  Heart transplanted patients

 -  Active hepatitis B or C or HIV patients

 -  Any contra-indication for coronary angiography.