Abstract
Background
Late thrombotic events are important complications associated with intracoronary brachytherapy (ICBT) using ionizing radiation (IR) or with antiproliferative treatment modalities such as drug-eluting stents (DES). The mechanism mediating these thrombotic events is not well understood. This study assessed the effect of prolonged clopidogrel treatment on tissue factor (TF) expression in coronary arteries and on the circulating TF level after percutaneous transluminal coronary angioplasty /ICBT in a porcine coronary model.
Methods
Pigs were treated with aspirin plus a 300 mg loading dose of clopidogrel one day before percutaneous coronary intervention (PCI), followed by a daily dose of clopidogrel and aspirin. During PCI one of the two balloon-injured arteries was treated by brachytherapy. Animals were sacrificed at different time points. The pigs, which were sacrificed 3 months post-PCI, were divided into two groups (Group I: clopidogrel for 3 months; Group II: clopidogrel for 1 month). Plasma TF was measured by enzyme-linked immunosorbent assay in blood samples taken from all pigs before and immediately after intervention and before sacrifice. Morphometric analysis was performed on digitalized images employing the software LUCIA G for TF staining. Vascular TF expression levels were assessed by quantitative real-time polymerase chain reaction.
Results
Prolonged clopidogrel application significantly reduced coronary TF at the protein (Group I vs. II, 8.975±3.947% vs. 26.44±5.375%, P =.007) and mRNA level [Group I vs. II, (0.3501±0.0519)×10 −3 vs. (0.7073±0.0436)×10 −3 , P <.0005]. Circulating TF protein tended to be lower after 3 months than after 1 month clopidogrel treatment post-PCI (Group I vs. Group II, 488.3±35.37 pg/ml vs. 572.3±39.9 pg/ml, P =.130).
Conclusions
Prolonged clopidogrel treatment reduced coronary TF expression and tended to reduce the blood TF level post-PCI, thus possibly modulating the risk of late thrombosis.
1
Introduction
Application of ionizing radiation (IR) involves an increased risk of late thrombotic events (>1 month) with a reported incidence of 2.5–9.6% after intracoronary brachytherapy (ICBT) . Late thrombotic events are also supposed to be responsible for the increased mortality observed after antiproliferative therapies such as implantation of drug eluting stents (DES) for primary coronary lesions . The mechanisms mediating thrombotic events are not well understood. Several studies have analyzed the effects of IR on endothelial thrombogenic and thrombolytic function. These studies reported increased activation of the tissue factor (TF) pathway, followed by increased thrombotic events after IR . TF is a membrane-bound glycoprotein that induces thrombosis by activating factor VII (FVII), which in turn activates FIX and FX. FXa together with its cofactor FVa converts prothrombin to thrombin, and thrombin generation results in fibrin formation, platelet activation and thrombus formation .
Application of clopidogrel before and after percutaneous coronary intervention (PCI) has been found to reduce myocardial infarction and mortality . By inhibiting an adenosine diphosphate receptor (P2Y 12 receptor), thienopyridines attenuate platelet degranulation and the expression of multiple platelet surface adhesion molecules involved in platelet interactions with different cells .
Clopidogrel was shown to significantly reduce the blood TF concentration in patients with stable coronary artery disease (CAD) . TF is known to be present in the α-granules of platelets . By reducing platelet degranulation clopidogrel may reduce the platelet release of TF and in the platelet-mediated TF induction in various vascular cells , thus diminishing the blood TF concentration as reported in stable CAD patients .
The aim of our study was to examine the effects of PCI on TF expression in coronary arteries and on the circulating TF level in relation to the duration of clopidogrel treatment in a porcine model.
2
Materials and methods
2.1
Study design
All studies were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 85-23, revised 1996). 50 domestic crossbred pigs (weight 22−27 kg) received antiplatelet therapy including aspirin (325 mg/d) and clopidogrel (300 mg loading dose; 75 mg/d), starting the day before intervention. Two pigs died during the percutaneous transluminal coronary angioplasty (PTCA) procedure and were not used for statistical analysis, thus the data of 48 pigs were included in this study. Aspirin was continued until sacrifice in all animals. Animals were sacrificed by an overdose of potassium chloride after 24 h ( n =12), 1 month ( n =12) and 3 months postintervention ( n =24). The pigs, which were sacrificed 3 months postintervention, were divided into two groups (Group I and II). The first group (Group I, n =12) received clopidogrel in addition to aspirin for 3 months up to the time point of sacrifice. The second group (Group II, n =12) was treated with clopidogrel for only 1 month after PCI. Aspirin was continued for the whole study period up to 3 months ( Fig. 1 , study flow chart). Following a previously described protocol, cardiac catheterization was performed using a femoral artery approach according to standard procedures. All animals received periprocedural heparin. In each pig, PTCA was performed in two major coronary arteries by three times inflation of standard balloons, 20 mm in length and 3.5 or 4 mm in diameter, depending on angiographically evaluated vessel size (to ensure a balloon-to-artery ratio of 1.3–1.5:1, inflation duration: 30 s). One of the two balloon-injured arteries was randomly assigned to receive immediate radiation treatment with a beta-emitting source train. A noncentred Beta-Rail delivery catheter (40 mm, ø 2.13 mm; Novoste, Norcross, GA, USA) was introduced over a flexible 0.014-inch guidewire (Wizdom ST Floppy, Cordis, Miami, FL, USA) and positioned at the angioplasty site, ensuring a proximal and distal overhang. The delivery catheter was afterloaded with a 40 mm source train of 16 90 Sr/Y seeds (Novoste Beta-Cath). Dwell times were adapted to the applied balloon size to deliver 19.9−22.2 Gy to a depth of 2 mm from the centre of the source. The third major coronary artery remained untreated to serve as an uninjured control.
2.2
Histopathologic processing
Immediately after sacrifice, porcine hearts were harvested and perfusion fixed with 4% paraformaldehyde at 100 mm Hg via pressure tubing placed in the ascending aorta. The angioplasty site (PTCA), the angioplasty+brachytherapy site (PTCA+ICBT), and the corresponding uninjured major coronary artery (uninjured control) were dissected from each heart, subdivided into multiple segments, and embedded in paraffin.
2.3
Immunohistochemistry for TF and fibrin/fibrinogen
The TF and fibrin/fibrinogen staining was performed on 5-μm cross-sections from paraffin-embedded tissue blocks. Several slides made from every block were deparaffinised overnight by incubation with Roti-Histol (Roth, Karlsruhe, Germany). After rehydration, they were incubated in trypsin (1 mg/ml, Sigma, Munich, Germany) for 10 min at 37°C to enhance the penetration with the antibodies. The slides were washed twice in Tris-buffered saline (TBS) and blocked with Avidin/Biotin Blocking Kit according to manufacturer’s protocol (Vector Laboratories, Burlingame, CA, USA). Then the slides were incubated either with polyclonal rabbit anti-TF antibody (kindly provided by Nemerson Y., Mount Sinai School of Medicine, New York, NY, USA) or with polyclonal rabbit anti-fibrinogen antibody (DakoCytomation, Hamburg, Germany), both diluted 1:150 in TBS/fetal calf serum, overnight at 4°C. Negative controls were performed without the use of primary antibodies or using negative control IgG antibodies. After incubation with primary antibody at least, specimens were washed, incubated with an appropriate biotinylated secondary antibody (DakoCytomation, Hamburg, Germany), and counterstained with haematoxylin (Merck, Darmstadt, Germany). For detection, carbazole (AEC Substrate Kit, DAKO Cytomation, Hamburg, Germany) was used according to manufacturer’s instructions.
2.4
Morphometric analysis
Morphometric analysis was performed on digitalized images employing the software LUCIA G (Version 3.52ab, Nikon, Dusseldorf, Germany). The percentage of the positive stained area was measured on the total arterial area (interna und media of the arterial wall).
2.5
Tissue factor enzyme-linked immunosorbent assay
Blood was taken from all pigs before intervention, immediately postintervention and before sacrifice. Plasma TF was measured by an enzyme-linked immunosorbent assay (ELISA) assay, using a matched-pair antibody set, as described by the manufacturer’s instructions (Imubind Tissue Factor ELISA Kit, American Diagnostica, Pfingstadt, Germany).
2.6
RNA isolation
Total RNA of porcine coronaries was isolated with TRIzol (Invitrogen, Karlsruhe, Germany) according to manufacturer’s instruction, reverse-transcribed, and analysed by polymerase chain reaction (PCR) using primers listed in Table 1 . RNA was separated by electrophoresis on 1% agarose; integrity was checked electrophoretically and quantified spectrophotometrically.
| Amplicons (bp) | ||
|---|---|---|
| A | Probe sequences | |
| porcTFT-fwd2: 5′-GTC CCG CCT GTG TCT CTG TA-3′ | 94 | |
| porcTFT-rev2: 5′-GAG ACC GTC ACG CCA CAT T-3′ | ||
| pGAPDH T fwd: 5′-CAG CAA TGC CTC CTG TAC CA-3′ | 70 | |
| pGAPDH T rev: 5′-GAT GCC GAA GTT GTC ATG GA-3′ | ||
| B | Primer sequences | |
| porcTF so: 5′-FAM-TGC AGG AAG GAG CGG GCA GGT XT-PH-3′ | ||
| pGAPDH So: 5′-FAM-AAC TGC TTG GCA CCC CTG GCC XT-PH-3′ | ||
| C | Conditions | |
| Primer sequences (D), conditions and performance of conventional RT-PCR (cDNA standards for real time PCR) pTF: initial denaturation: 94°C, 2 min, 94°C, 1 min, 59°C, 1 min; 72°C, 90 s for 30 cycles; final extension: 72°C, 7 min pGAPDH: initial denaturation: 94°C, 2 min, 94°C, 30 s, 60°C, 30 s; 72°C, 90 s for 30 cycles; final extension: 72°C, 5 min | ||
| D | Primer of conventional PCR | |
| pTF st-fwd 1: 5′-TTT GCG GTC ATC GTC TTC GT-3′ | 440 | |
| pTFst-rev1: 5′-GGT TTT CCA GGG CAT TTC TT-3′ | ||
| pGAPDH stfwd: 5′-ATG CTG GTG CTG AGT ATG TC-3′ | 245 | |
| pGAPDHTREV 5′-GAT GCC GAA GTT GTC ATG GA-3′ | ||
2.7
Real-time PCR (TaqMan)
First, 1 μg total RNA of each sample was transcribed into complementary deoxyribonucleic acid (cDNA) by using avian myeoblastosis virus reverse transcriptase and random hexamer primers according to the supplier’s instructions (Roche Applied Sciences, Mannheim, Germany). No reverse transciptase controls were performed to exclude amplification of genomic DNA. Then, 5 μl of each complementary DNA preparation was diluted to a final PCR volume of 25 μl containing 12.5 μl TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), and 300 nM primers (TIB Molbiol, Berlin, Germany) and 100 nM probes as listed in Table 1 A. For generation of standards for real-time PCR TF and glyceraldehyde −3 -phosphate dehydrogenase (GAPDH) products were generated by conventional PCR. Five μl of each cDNA was diluted to a volume of 50 μl PCR mix with 5 μl 10× buffer (1.5 mM MgCl 2 ), 5 μl dNTP (2 mM), 0.5 μl (2.5 U/μl) Taq polymerase (all reagents from Roche Diagnostics, Mannheim, Germany), 2.5 μl (0.5 μg/μl) porcine TF (pTF)st-fwd1 primer, 2.5 μl (0.5 μg/μl) pTFst-rev1 primer; 2.5 μl (0,5 μg/μl) porcine GAPDH (pGAPDH)st-fwd primer, 2.5 μl (0.5 μg/μl) pGAPDH T-rev primer (TIB MolBiol, Berlin, Germany) ( Table 1 B). Real-time PCR was performed using ABI Prism 7000 Sequence Detection System (Applied Biosystems) under the following conditions: 50°C, 2 min; 35°C, 10 min; 40 cycles 95°C, 15 s; 60°C, 1 min. The generated cDNAs were serially diluted in PCR grade water. A standard curve with 5 cDNA dilutions in triplicates of porcine TF (10 4 , 10 3 , 10 2 , 10 1 , 10 0 copies/μl) and porcine GAPDH (10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies/μl) cDNA was included in each respective PCR run. As an internal standard, GAPDH cDNA was measured analogous to tissue factor.
2.8
Statistical analysis
SPSS statistical software version 11.0.1 was used for statistical analysis. Quantitative data were expressed as mean±S.E.M.; P ≤.05 was considered significant. The data were tested for normal distribution using Kolmogoroff Smirnov test. Then, all data were tested using analysis of variance to evaluate overall differences between the time points in a group and between the different groups. If the test revealed significant differences between the time points or groups pairwise comparisons were conducted using the t test for independent or dependent samples.
2
Materials and methods
2.1
Study design
All studies were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 85-23, revised 1996). 50 domestic crossbred pigs (weight 22−27 kg) received antiplatelet therapy including aspirin (325 mg/d) and clopidogrel (300 mg loading dose; 75 mg/d), starting the day before intervention. Two pigs died during the percutaneous transluminal coronary angioplasty (PTCA) procedure and were not used for statistical analysis, thus the data of 48 pigs were included in this study. Aspirin was continued until sacrifice in all animals. Animals were sacrificed by an overdose of potassium chloride after 24 h ( n =12), 1 month ( n =12) and 3 months postintervention ( n =24). The pigs, which were sacrificed 3 months postintervention, were divided into two groups (Group I and II). The first group (Group I, n =12) received clopidogrel in addition to aspirin for 3 months up to the time point of sacrifice. The second group (Group II, n =12) was treated with clopidogrel for only 1 month after PCI. Aspirin was continued for the whole study period up to 3 months ( Fig. 1 , study flow chart). Following a previously described protocol, cardiac catheterization was performed using a femoral artery approach according to standard procedures. All animals received periprocedural heparin. In each pig, PTCA was performed in two major coronary arteries by three times inflation of standard balloons, 20 mm in length and 3.5 or 4 mm in diameter, depending on angiographically evaluated vessel size (to ensure a balloon-to-artery ratio of 1.3–1.5:1, inflation duration: 30 s). One of the two balloon-injured arteries was randomly assigned to receive immediate radiation treatment with a beta-emitting source train. A noncentred Beta-Rail delivery catheter (40 mm, ø 2.13 mm; Novoste, Norcross, GA, USA) was introduced over a flexible 0.014-inch guidewire (Wizdom ST Floppy, Cordis, Miami, FL, USA) and positioned at the angioplasty site, ensuring a proximal and distal overhang. The delivery catheter was afterloaded with a 40 mm source train of 16 90 Sr/Y seeds (Novoste Beta-Cath). Dwell times were adapted to the applied balloon size to deliver 19.9−22.2 Gy to a depth of 2 mm from the centre of the source. The third major coronary artery remained untreated to serve as an uninjured control.
