The study design, patients demographics and inclusion and exclusion criteria have been published in detail previously . Briefly, one hundred patients with acute anterior wall ST-elevation myocardial infarction, both gender, age between 40 and 75 years were included at Ullevål University Hospital and Rikshospitalet University Hospital, Oslo, Norway. All the patients participated in the ASTAMI (Autologous Stem cell Transplantation in Acute Myocardial Infarction) trial with the culprit lesion located in the left anterior descending artery (LAD), proximal to the 2. diagonal branch, and were treated successfully with PCI and stent implantation within 2 – 12 hours from symptom start, in addition to dual antiplatelet therapy . The inclusion criteria were peak creatine kinase (CK) MB above 3 times the upper reference level and hypo- or akinesia in more than 2 of 16 segments of the left ventricle determined by echocardiography. Patients with cardiogenic shock, previous Q-wave infarction and considerable co-morbidity with short life expectancy were excluded from the study. The investigation conformed with the principles outlined in the Declaration of Helsinki. The Regional Committee for Medical Research Ethics approved the study protocol, and written informed consent was obtained from all the patients. The present study is a substudy of the ASTAMI trial that is registered at http://www.clinicaltrials.gov, NCT 00199823.
The included patients were randomized 1:1 to receive intracoronary injections of autologous mononuclear bone marrow cells (mBMC) or to a control group without any further interventions. The mBMC group was aspirated for 50 ml bone marrow from the iliac crest in local anesthesia 4 – 7 days after the acute PCI and the next day, a median of 6 days after the AMI, they received intracoronary injections of mBMC in the LAD. All the patients were treated with a loading dose of clopidogrel 300 mg and thereafter 75 mg daily in addition to an initial dose of aspirin 300 mg followed by 75 mg daily. Patients with LV thrombus were treated with low molecular weight heparin and further warfarin with a target international normalized ratio of 2.0 to 2.5 for at least 3–6 months. Fasting blood samples were drawn 4–5 days (baseline), 6–7 days, 8–9 days, 2–3 weeks and 3 months after the AMI for determination of circulating haemostatic markers and selected inflammatory mediators (only at baseline). In accordance with the study protocol a screening echocardiographic examination of all eligible patients was performed within the first 1–3 days after the acute PCI. Thereafter, echocardiograms were obtained by Vivid 7 scanner (GE Vingmed Ultrasound, Horten, Norway) of all the included patients 4.5 ± 1.1 days after the acute AMI and repeated after 3 months. Additional echocardiographic examinations were performed in between the predefined time points when clinically indicated. Magnetic resonance imaging (MRI) was performed with a 1.5-tesla scanner (Siemens, Germany) 18.8 ± 4.3 days after the acute PCI. Infarct size of the LAD area, in percent, was obtained by electrocardiogram-gated single photon emission computed tomography (SPECT) (GE Medical Systems with 4D-MSPECT software) 4.0 ± 1.4 days after the AMI.
Soluble tissue factor (TF) was measured by an enzyme-linked immunoassay (Imubind® Tissue Factor, American Diagnostica Inc., US), prothrombin fragment 1+2 (F1+2) by an enzyme immunoassay (Enzygnost®F1+2, Siemens Healthcare Diagnostics, Marburg, Germany). Endogenous thrombin potential (ETP) was determined according to the manufacturer’s instruction (Thrombinoscope BV, Maastricht, The Netherlands) and the thrombin generation was measured on the Fluoroscan Ascent® fluorometer (Thermo Fisher Scientific OY, Vantaa, Finland). A reagent of rTF and phospholipids in addition to a thrombin specific fluorogenic substrate (Z-Gly-Gly-Arg-AMC, Bachem, Bubendorf, Switzerland) in Hepes buffer containg CaCl2, were added to the plasma prior to start to obtain a final concentration of 5 pM, 4 uM and 2.5 mM, respectively. In order to calculate the final results, plasma was analyzed along with a thrombin calibrator with known thrombin activity as a reference. The software program (Thrombinoscope BV, version 184.108.40.206) enabled the calculation of ETP. All the experiments were run in duplicates. D-dimer was measured by enzyme immunoassay (Asserachrom, D-di™, Stago Diagnostica, Asniere, France) and plasminogen activator inhibitor 1 (PAI-1) activity by a bio immunoassay (Trinilize PAI-1 activity, Trinity Biotech, plc, Bray, Co. Wicklow, Ireland). The levels of tissue plasminogen activator (tPA) antigen were determined by enzyme immunoassay (TintElize®tPA, Trinity Biotech, Jamestown, NY, US). Tumor necrosis factor α (TNFα) and interleukin 6 (IL-6) were measured by enzyme immunoassays obtained from R&D Systems Europe (Abingdon, Oxon, UK). C-Reactive protein (CRP) was determined by an enzyme-linked immunosorbent assay (DRG Instruments GmbH, Germany) (detection limit 0.1 mg/L). In our laboratory, the interassay coefficient of variation for TF was 4.4%; F1+2 7.8%; ETP 2.7%; D-dimer 6.5%; PAI-1 4.4%; tPA 3.5%; TNFα 8,5%; IL-6 10.7%; CRP<5%.
Total RNA was extracted from PAXgene® Blood RNA tubes in a subset of patients with (n=7) and without (n=10) LV thrombus. The PAXgene® Blood RNA Kit (PreAnalytix, Qiagen GmbH, Germany), including an extra cleaning step (RNeasy®MinElute® Cleanup Kit, Qiagen) was used. Total RNA was reversely transcribed in a total volume of 20 μl, using the Omniscript® RT Kit (Qiagen), Oligos (dTs) and Rnase Inhibitor (Applied Biosystems, Foster City, CA, USA). TF mRNA levels were determined by real-time PCR on the ABI Prism 7900 HT Sequence Detection System, including the TaqMan® Gene Expression TF Assay (Hs01076029_m1). The TF mRNA levels were normalized to β-2-microglobulin (Hs99999907_m1, Applied Biosystems) and fold expression (relative quantification using the ΔΔCt method) was determined in relation to a reference sample, as previously described .
Variables are expressed as proportions, medians with 25,75 percentiles or means with standard deviation as appropriate. Differences between groups were analysed with the Mann–Whitney test for continuous variables. Categorical data were analyzed by the chi-square test. Friedman test was applied for testing the intragroup differences between several related samples and Wilcoxon test was used when Friedman test was significant, to assess within group changes from baseline to the subsequent time points. A trend analysis in quartiles of variables was performed by the chi square test to estimate the cut off levels in relation to LV thrombus. Logistic regression analyses with LV thrombus as the dependent variable were performed with adjustments for infarct size. Correlation analysis was done with Spearmans rho. All tests were two-sided, and p values <0.05 were considered statistically significant. PASW software package version 18.0 for Mac OS X was used for data analyses.