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Fibrin clot formation and fibrinolysis in patients with a history of coronary stent thrombosis
Thrombosis Research 143 (2016) 58–62
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Fibrin clot formation and fibrinolysis in patients with a history of coronary stent thrombosis Thea C. Godschalk a,⁎,1, Joke Konings b,1,2, José W. Govers-Riemslag b, Jurriën M. ten Berg a, Christian M. Hackeng c, Hugo ten Cate b a
St Antonius Center for Platelet Function Research, Department of Cardiology, St. Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, The Netherlands Laboratory for Clinical Thrombosis and Haemostasis, Departments of Internal Medicine and Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands c St Antonius Center for Platelet Function Research, Department of Clinical Chemistry, St. Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, The Netherlands b
a r t i c l e
i n f o
Article history: Received 8 March 2016 Received in revised form 29 April 2016 Accepted 30 April 2016 Available online 3 May 2016 Keywords: Coronary thrombosis Fibrin Fibrinolysis Percutaneous coronary intervention Stent
a b s t r a c t Introduction: Coronary stent thrombosis is a devastating complication of percutaneous coronary intervention (PCI). Multiple factors underlie the pathophysiological mechanisms of stent thrombosis. Previous studies demonstrated that patients with stent thrombosis, compared to control PCI patients, formed denser fibrin clots in vitro which were more resistant to fibrinolysis, suggesting that altered fibrin clot properties may contribute to the pathophysiology of stent thrombosis. We assessed the plasma fibrin clot formation and fibrinolysis of patients with and without stent thrombosis. Methods: Cases (patients with stent thrombosis) and matched controls (patients without stent thrombosis) were included for a matched case-control study. Matching was performed on indication and time of the index PCI (initial stent implantation) from the cases. Fibrin clot formation and fibrinolysis were assessed in vitro by turbidimetric assays, with human thrombin to initiate fibrin polymerization and tissue type plasminogen activator to initiate fibrinolysis. Lag time, maximal absorbance and clot lysis time were determined by these assays. Results: In total, 27 cases and 27 controls were included. No significant differences were observed between cases and controls in lag time (173 vs. 162 s, p = 0.18), maximal absorbance (0.78 vs. 0.83, p = 0.36), and clot lysis time (69 vs. 71 min, p = 0.78). Fibrin clot formation and fibrinolysis were not associated with stent thrombosis. Conclusions: Plasma fibrin clot formation and fibrinolysis were not significantly different between patients with stent thrombosis and matched control patients, suggesting that fibrin clot formation and fibrinolysis play no significant role in the pathophysiology of stent thrombosis. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Coronary stent thrombosis is a feared complication of percutaneous coronary intervention (PCI). Around 80% of the patients with stent thrombosis present with a myocardial infarction [1,2], and stent thrombosis is associated with mortality rates between 10 and 40% [3–5]. The incidence of stent thrombosis is approximately 1–4%, despite Abbreviations: BMS, bare metal stent; CI, confidence interval; DES, drug eluting stent; OR, odds ratio; PCI, percutaneous coronary intervention. ⁎ Corresponding author at: St. Antonius Hospital, P.O. Box 2500, 3430 EM Nieuwegein, The Netherlands. E-mail addresses: [email protected] (T.C. Godschalk), [email protected] (J. Konings), [email protected] (J.W. Govers-Riemslag), [email protected] (J.M. ten Berg), [email protected] (C.M. Hackeng), [email protected] (H. ten Cate). 1 These authors contributed equally to this project. 2 Synapse Research Institute, Cardiovascular Research Institute Maastricht, Maastricht University, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands.
http://dx.doi.org/10.1016/j.thromres.2016.04.026 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
dual antiplatelet therapy with aspirin and clopidogrel [3,6]. Multiple factors underlie the pathophysiological mechanisms of stent thrombosis, such as stent underexpansion and high residual platelet reactivity [7,8]. Thrombus formation includes the interplay of platelet activation and aggregation with fibrin clot formation. Clot stability is largely determined by mechanical strength and sensitivity to fibrinolysis. Previous studies by Undas and co-workers [9,10] demonstrated that patients with stent thrombosis, compared to control PCI patients, formed denser fibrin clots which were more resistant to fibrinolysis. In these studies, 90% of the patients received a bare metal stent (BMS). However, the use of drug eluting stents (DES) compared to BMS has increased in recent years, which might have changed the risk factors associated with stent thrombosis. Therefore, we assessed the potential of plasma fibrin clot formation (lag time, maximal absorbance) and fibrinolysis (clot lysis time) from patients with a history of stent thrombosis compared to matched control patients in a currently representative cohort.
T.C. Godschalk et al. / Thrombosis Research 143 (2016) 58–62
2. Patients and methods 2.1. Study design and population A single-center case-control study including PCI patients with stent implantation was performed. Cases underwent an index PCI (PCI of initial stent implantation) after which they suffered from a definite stent thrombosis according to the Academic Research Consortium criteria [11]. The timing of stent thrombosis was divided into cases with early (≤ 30 days after index PCI) and late stent thrombosis (b 30 days after index PCI). Included cases had suffered from stent thrombosis between January 2007 and September 2011. Controls underwent an index PCI without suffering from stent thrombosis between index PCI and blood sampling. Control patients were matched 1:1 based on the indication (stable angina pectoris, unstable angina pectoris/non ST-segment elevation myocardial infarction, ST-segment elevation myocardial infarction) and time (± 14 days) of the index PCI from the cases. Subjects using oral anticoagulants or heparins at the time of blood collection were excluded. We selected all patients with stent thrombosis, who were matched with a control patient, and with available blood samples from the matched stent thrombosis and control patients. Written informed consent was provided by all participants. The study was approved by the local institutional Ethics Committee and was conducted according to the principles of the Declaration of Helsinki. 2.2. Blood collection and preparation All subjects had visited the St Antonius Hospital for blood sampling (Nieuwegein, The Netherlands). The minimal time interval between the last performed PCI and blood sampling was one month for cases and controls. Venous blood samples were collected from the antecubital vein using 21-gauge needles and Vacuette® tubes (Greiner Bio-one, Frickenhausen, Germany) containing 3.2% (w/v) sodium citrate. To avoid haemostatic activation, the first 5 ml of free-flowing blood was discarded. Platelet poor plasma was obtained by two separate centrifugation steps. Samples were first centrifuged for 10 min at 150g, followed by 15 min at 2500g. All platelet poor plasma samples were stored at −80 °C until analysis. 2.3. Fibrin clot formation and fibrinolysis assay To monitor fibrin clot formation, plasma samples were diluted 1.67 times with Hepes-buffer (25 mM Hepes (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid), 150 mM NaCl, pH = 7.5) and 125 μl of diluted plasma was transferred into a low binding polystyrene 96-well plate (Greiner, Frickenhausen, Germany). Fibrin polymerization was started by addition of 25 μl activation mixture. The activation mixture contained human thrombin (Enzyme Research Laboratories, Swansea, UK; final concentration: 0.75 nmol/l), phospholipids which were prepared by sonication as described earlier [12] (1,2-dioleoyl-sn-glycero3-phosphoserine, 1,2-dioeoyl-sn-glycero-3-phosphocholine, 1,2dioleoyl-snglycero-3-phosphoethanolamine (DOPS/DOPC/DOPE, 20/ 60/20, mol/mol/mol), Avanti Polar lipids Inc., Alabaster, Alabama, USA; final concentration: 10 μmol/l), and CaCl2 (final concentration: 16 mmol/l). Measurements were at 405 nm every 15 s for 60 min at 37 °C using an ELx808 plate reader (Biotek Instruments, Winooski, VT). The lag time, defined as the time to an increase of 0.01 optical density from baseline, together with the maximal absorbance were determined from the curves of the turbidity measurements. Turbidity measurements were performed in duplicate. To monitor fibrinolysis, recombinant tissue plasminogen activator (Boehringer Ingelheim, Alkmaar, the Netherlands; final concentration: 50 ng/ml) was added to the activation mixture, as described as above, at the start of the assay and turbidity was recorded for 6 h. Clot lysis
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time was calculated as the time from 50% clot formation to 50% fibrinolysis. Lysis measurements were performed in triplicate. The final concentrations of human thrombin and recombinant tissue plasminogen activator used in these assays were determined by turbidimetric assays with a normal plasma pool of healthy volunteers not using any medication. 2.4. Laboratory measurements Plasma concentrations of fibrinogen were measured using a Sysmex® CA-7000 System Automated Coagulation Analyzer with reagents obtained from Siemens Healthcare Diagnostics (Marburg, Germany) according to the Claus method [13]. Platelet count was measured using a LH 750 (Beckman Coulter) and cholesterol levels were using a Cobas 6000 (Roche Diagnostics). Hypercholesterolemia was defined as an increased level of total cholesterol (N 5.0 mmol/l), triglycerides (N 1.5 mmol/l), LDL (N 2.5 mmol/l) or a decreased level of HDL (b 1.0 mmol/l). 2.5. Statistical analysis Statistical analyses were performed with PRISM for Windows, version 5.00 (GraphPad Software, San Diego, CA, USA), and SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). Continuous data are expressed as mean ± standard deviation, and categorical data are expressed as frequencies no./total no. (%). Baseline characteristics and laboratory measurements at the time of blood sampling were analyzed using McNemar test or paired Student's t-test. Differences between cases and controls for fibrin clot properties were analyzed using univariate conditional logistic regression, reported as odds ratios (OR) with 95% confidence intervals (CI). Subgroup analysis was performed for cases with early vs. late stent thrombosis, and for cases with early stent thrombosis and their matched controls, as well as for cases with late stent thrombosis and their matched controls. A two-tailed p-value b 0.05 was considered as statistically significant. 3. Results 3.1. Patients A total of 27 cases and 27 controls were included. Twelve cases had experienced an early stent thrombosis and 15 cases a late stent thrombosis. None of the matched controls experienced a stent thrombosis between index PCI and blood sampling. Baseline characteristics are summarized in Table 1. Compared to controls, significantly more cases were current smokers at time of index PCI. The rate of hypertension was numerically higher for cases than for controls however without reaching statistical significance. The mean time-interval between stent thrombosis and blood sampling for cases was 35 ± 25 months and between index PCI and blood sampling for controls was 42 ± 26 months. More cases than controls were on dual antiplatelet therapy at the time of blood sampling (Table 2). Fibrinogen levels, cholesterol levels and platelet count were comparable between cases and matched controls. 3.2. Fibrin clot properties The fibrin clot properties were not significantly different between cases and controls (lag time: 173 ± 47 s vs. 162 ± 28 s, p = 0.18; maximal absorbance: 0.78 ± 0.16 vs. 0.83 ± 0.21, p = 0.36; clot lysis time: 69 ± 20 min vs. 71 ± 25 min, p = 0.78) (Fig. 1). Lag time, maximal absorbance, and clot lysis time were not associated with stent thrombosis (Table 3). Subgroup analyses for cases with early vs. late stent thrombosis showed no significant differences in fibrin clot properties (Table 4). Also, analyses of cases with early
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Fibrinogen levels were correlated with maximal absorbance for cases and controls (r = 0.91 and r = 0.91, both p b 0.001).
Table 1 Baseline characteristics at the time of index PCI.
Clinical characteristics Female Age (years) Body mass index (kg/m2) Current smoking Diabetes mellitus Hypertension Hypercholesterolemia Family history of CVD Medical history Prior MI Prior PCI Prior CABG Malignancy Renal failure PAD AF Thrombo-embolic events LVEF b 45% Indicationa Stable angina pectoris UAP/NSTEMI STEMI Procedural characteristics Bare metal stents Drug eluting stents Target vessel RCA LAD RCX Total stent length (mm) Antiplatelet therapy GP IIb/IIIa Aspirin Clopidogrel
Cases (n = 27) n (%)
Matched controls (n = 27) n (%)
p-Value
4 (14.8) 57 ± 11 26.3 ± 3.2 18 (72.0) 6 (22.2) 13 (48.1) 15 (60.0) 19 (73.1)
3 (11.1) 60 ± 9 26.9 ± 3.4 8 (32.0) 3 (11.1) 5 (18.5) 12 (48.0) 14 (53.8)
1.000 0.240 0.494 0.002 0.453 0.077 0.581 0.267
9 (33.3) 7 (25.9) 1 (3.7) 1 (3.7) 1 (3.8) 3 (11.1) 0 (0.0) 3 (11.1) 6 (37.5)
5 (18.5) 8 (29.6) 1 (3.7) 3 (11.1) 1 (3.8) 0 (0.0) 1 (3.7) 1 (3.7) 4 (25.0)
0.344 1.000 1.000 0.625 1.000 0.250 1.000 0.625 0.625
9 (33.3) 4 (14.8) 14 (51.9)
9 (33.3) 4 (14.8) 14 (51.9)
1.000 1.000 1.000
9 (33.3) 18 (66.7)
11 (40.7) 16 (59.3)
0.754 0.754
9/2 (33.3) 16 (59.3) 2 (7.4) 27.0 ± 13.6
11 (40.7) 9 (33.3) 7 (25.9) 23.3 ± 10.5
0.791 0.065 0.180 0.268
8 (29.6) 24 (96.0) 23 (92.0)
6 (22.2) 24 (96.0) 22 (88.0)
0.754 1.000 1.000
4. Discussion
PCI: percutaneous coronary intervention; BMI: body mass index; CVD: cardiovascular disease; MI: myocardial infarction; CABG: coronary artery bypass graft; PAD: peripheral artery disease; AF: atrial fibrillation; LVEF: left ventricular ejection fraction; UAP: unstable angina pectoris; NSTEMI: non-ST-segment elevation myocardial infarction; STEMI: STsegment elevation myocardial infarction; RCA: right coronary artery; LAD: left anterior descending artery; RCX: right circumflex artery; GP: glycoprotein. Continuous data are presented as mean ± standard deviation. a Matched item.
stent thrombosis vs. their matched controls and cases with late stent thrombosis vs. their matched controls showed no significant differences in fibrin clot properties (Table 5).
Table 2 Characteristics at the time of blood sampling.
Medication Aspirin DAPT Clopidogrel Prasugrel Ticagrelor Statins Laboratory measurements Fibrinogen (g/l) Platelet count (103/μl) Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l)
Cases (n = 27) n (%)
Matched controls (n = 27) n (%)
p-Value
27 (100.0) 20 (74.1) 13 (48.1) 5 (18.5) 2 (7.4) 27 (100)
27 (100.0) 10 (37.0) 8 (29.6) 2 (7.4) 0 (0.0) 25 (92.6)
NA 0.01 0.27 0.38 0.50 0.50
3.6 ± 0.7 225 ± 42 4.1 ± 0.8 2.1 ± 0.6 1.28 ± 0.83 1.8 ± 0.7
3.6 ± 0.7 211 ± 51 4.1 ± 0.8 1.9 ± 0.6 1.20 ± 0.33 2.1 ± 1.3
0.88 0.34 0.97 0.12 0.71 0.33
NA: not applicable; DAPT: dual antiplatelet therapy; LDL: low density lipoprotein; HDL: high density lipoprotein. Continuous data are presented as mean ± standard deviation.
This study did not observe differences between in vitro fibrin clot formation and fibrinolysis in patients with a history of stent thrombosis and matched control PCI patients, suggesting that fibrin clot properties are not altered in patients with a history of stent thrombosis. Moreover, in the pathophysiology of stent thrombosis, plasma components play no important role in fibrin clot formation and fibrinolytic susceptibility. Our results are contrary to earlier observations of in vitro experiments [9,10]. Undas et al. showed altered fibrin clot properties and an impaired susceptibility to fibrinolysis for stent thrombosis patients compared to control PCI patients [9]. Our study methods differ from this study at three points. First, the methods of fibrin clot formation and fibrinolysis were slightly different. The concentration of thrombin for the turbidimetric assays was lower in our study, with a more sensitive assay as a result. Second, we matched control patients based on the indication and time of the index PCI of the case, whereas in the study of Undas et al. control patients were matched for numerous parameters, which could not always be realized. Case-control matching based on age and sex might be a more common method, nevertheless, we are convinced that matching based on indication for index PCI is more important, because the pathophysiology and/or clinical outcome of ST-segment elevation myocardial infarction and e.g. stable angina pectoris is different, and will be more suitable to discover predictors for stent thrombosis. Matching based on index PCI resulted in an equal follow-up period and a PCI procedure around the same period of time regarding stent placement and balloon dilatation methods. Our matching method still resulted in an equal distribution for age and sex. Third, the ratio of patients included with early vs. late stent thrombosis was different, respectively 12 (44%) vs. 15 (56%) for our study and 41 (87%) vs. 6 (13%) for the study by Undas. Literature described an impaired fibrinolytic potential for patients with early stent thrombosis compared to control PCI patients [10]. It could be hypothesized that the formation of a denser fibrin clot and hypofibrinolysis might be more important for the pathophysiology of early stent thrombosis than for late stent thrombosis, as the underlying pathophysiological mechanisms for early and late stent thrombosis also show differences. For example, delayed vascular healing is predominantly associated with late stent thrombosis [14], while discontinuation of clopidogrel is especially an important risk factor for the occurrence of early stent thrombosis [8]. Our subgroup analysis did not reveal a difference in fibrin clot formation and fibrinolysis between cases with early or late stent thrombosis, however no firm conclusions could be drawn from these analyses as the number of patients in the subgroups was small. One of the main determinants of fibrin clot structure is the concentration of thrombin [15]. The amount of thrombin in our in vitro experiments was kept constant and we cannot exclude that there might be a risk in vivo in the stented area for increased thrombin formation, which in case of clot formation will contribute to a denser fibrin clot structure. At the time of index PCI, more cases than controls were current smokers and tended to have more often hypertension. Smoking and hypertension both have an effect on fibrin clot properties. Smoking increases the clot strength and the resistance to fibrinolysis [16,17]. The use of antihypertensive agents increases the permeability and reduces the resistance to fibrinolysis in patients with arterial hypertension [18]. Whether smoking behavior was persistent until blood sampling is unknown and cannot be correlated to the fibrin clot properties in this study. At the time of blood sampling, more cases than controls were on dual antiplatelet therapy, as dual antiplatelet therapy was continued
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Fig. 1. Fibrin clot formation and fibrinolysis of cases and controls. (A) lag time, (B) maximal absorbance (A405 nm), and (C) clot lysis time in stent thrombosis patients (cases, n = 27) and matched control patients (controls, n = 27). Whiskers display mean ± standard deviation.
Table 3 Unadjusted OR of fibrin clot properties for stent thrombosis.
Lag time (s) Maximal absorbance CLT (min)
OR (95% CI)
p value
1.01 (0.99–1.03) 0.24 (0.01–4.97) 1.00 (0.98–1.02)
0.21 0.36 0.77
5. Conclusions
OR: odds ratio; CI: confidence interval; CLT: clot lysis time. Data are expressed as mean ± standard deviation.
Table 4 Fibrin clot properties of cases with early vs. late stent thrombosis. Early ST (n = 12)
Late ST (n = 15)
OR (95% CI)
measurements of individual fibrinolytic components. The causes of stent thrombosis were not included, which can be variable and are often unknown.
In conclusion, no differences were observed in fibrin clot formation and fibrinolysis in plasma of patients with a history of stent thrombosis compared with matched control patients, suggesting that fibrin clot formation and fibrinolysis do not have a significant role in the pathophysiology of stent thrombosis.
p-Value
Conflicts of interest
Lag time (s) 168 ± 49 178 ± 46 1.01 (0.99–1.02) 0.59 Maximal absorbance 0.74 ± 0.16 0.82 ± 0.16 33.83 (0.18–6420.35) 0.19 CLT (min) 62 ± 14 75 ± 23 1.05 (0.99–1.11) 0.14
None.
ST: stent thrombosis; OR: odds ratio; CI: confidence interval; CLT: clot lysis time. Data are expressed as mean ± standard deviation.
beyond one year after PCI in patients with stent thrombosis. Studies showed contrary results in clopidogrel altering fibrinogen levels [19, 20], another main determinant of fibrin clot structure [15]. However, fibrinogen levels were not significantly different between cases and controls in our study. Also, cholesterol levels and statin and aspirin use, factors that are known for their influence on fibrin clot formation and lysis [21], were not significantly different between cases and controls. Our study is hampered by the relatively small sample size and the small amount of platelet poor plasma, which limited the laboratory
Contributors TCG and JK designed and conducted the study, analyzed and interpreted the data and wrote the manuscript. JWG, JMB, CMH and HC designed the study, interpreted the data, and critically reviewed the manuscript.
Acknowledgements JK was funded by the Netherlands Heart Foundation (grant 2008B120). TCG was funded by the FP7 program of the European Commission (grant 260309). The funding sources were not involved in the establishment of the study and writing of the article.
Table 5 Fibrin clot properties of cases with early or late stent thrombosis and their matched controls. Cases with early stent thrombosis vs. controls
Lag time (s) Maximal absorbance CLT (min)
Cases (n = 12)
Matched controls (n = 12)
OR (95% CI)
p-Value
168 ± 49 0.74 ± 0.16 62 ± 14
158 ± 25 0.84 ± 0.18 75 ± 31
1.01 (0.98–1.03) 0.06 (0.00–7.11) 0.97 (0.93–1.02)
0.51 0.25 0.27
Cases (n = 15)
Matched controls (n = 15)
OR (95% CI)
p-Value
178 ± 46 0.82 ± 0.16 75 ± 23
164 ± 30 0.83 ± 0.24 68 ± 20
1.02 (0.99–1.05) 0.75 (0.01–40.13) 1.01 (0.98–1.05)
0.26 0.89 0.43
Cases with late stent thrombosis vs. controls
Lag time (s) Maximal absorbance CLT (min)
OR: odds ratio; CI: confidence interval; CLT: clot lysis time. Data are expressed as mean ± standard deviation.
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Full Length Article
Fibrin clot formation and fibrinolysis in patients with a history of coronary stent thrombosis Thea C. Godschalk a,⁎,1, Joke Konings b,1,2, José W. Govers-Riemslag b, Jurriën M. ten Berg a, Christian M. Hackeng c, Hugo ten Cate b a
St Antonius Center for Platelet Function Research, Department of Cardiology, St. Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, The Netherlands Laboratory for Clinical Thrombosis and Haemostasis, Departments of Internal Medicine and Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands c St Antonius Center for Platelet Function Research, Department of Clinical Chemistry, St. Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, The Netherlands b
a r t i c l e
i n f o
Article history: Received 8 March 2016 Received in revised form 29 April 2016 Accepted 30 April 2016 Available online 3 May 2016 Keywords: Coronary thrombosis Fibrin Fibrinolysis Percutaneous coronary intervention Stent
a b s t r a c t Introduction: Coronary stent thrombosis is a devastating complication of percutaneous coronary intervention (PCI). Multiple factors underlie the pathophysiological mechanisms of stent thrombosis. Previous studies demonstrated that patients with stent thrombosis, compared to control PCI patients, formed denser fibrin clots in vitro which were more resistant to fibrinolysis, suggesting that altered fibrin clot properties may contribute to the pathophysiology of stent thrombosis. We assessed the plasma fibrin clot formation and fibrinolysis of patients with and without stent thrombosis. Methods: Cases (patients with stent thrombosis) and matched controls (patients without stent thrombosis) were included for a matched case-control study. Matching was performed on indication and time of the index PCI (initial stent implantation) from the cases. Fibrin clot formation and fibrinolysis were assessed in vitro by turbidimetric assays, with human thrombin to initiate fibrin polymerization and tissue type plasminogen activator to initiate fibrinolysis. Lag time, maximal absorbance and clot lysis time were determined by these assays. Results: In total, 27 cases and 27 controls were included. No significant differences were observed between cases and controls in lag time (173 vs. 162 s, p = 0.18), maximal absorbance (0.78 vs. 0.83, p = 0.36), and clot lysis time (69 vs. 71 min, p = 0.78). Fibrin clot formation and fibrinolysis were not associated with stent thrombosis. Conclusions: Plasma fibrin clot formation and fibrinolysis were not significantly different between patients with stent thrombosis and matched control patients, suggesting that fibrin clot formation and fibrinolysis play no significant role in the pathophysiology of stent thrombosis. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Coronary stent thrombosis is a feared complication of percutaneous coronary intervention (PCI). Around 80% of the patients with stent thrombosis present with a myocardial infarction [1,2], and stent thrombosis is associated with mortality rates between 10 and 40% [3–5]. The incidence of stent thrombosis is approximately 1–4%, despite Abbreviations: BMS, bare metal stent; CI, confidence interval; DES, drug eluting stent; OR, odds ratio; PCI, percutaneous coronary intervention. ⁎ Corresponding author at: St. Antonius Hospital, P.O. Box 2500, 3430 EM Nieuwegein, The Netherlands. E-mail addresses: [email protected] (T.C. Godschalk), [email protected] (J. Konings), [email protected] (J.W. Govers-Riemslag), [email protected] (J.M. ten Berg), [email protected] (C.M. Hackeng), [email protected] (H. ten Cate). 1 These authors contributed equally to this project. 2 Synapse Research Institute, Cardiovascular Research Institute Maastricht, Maastricht University, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands.
http://dx.doi.org/10.1016/j.thromres.2016.04.026 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
dual antiplatelet therapy with aspirin and clopidogrel [3,6]. Multiple factors underlie the pathophysiological mechanisms of stent thrombosis, such as stent underexpansion and high residual platelet reactivity [7,8]. Thrombus formation includes the interplay of platelet activation and aggregation with fibrin clot formation. Clot stability is largely determined by mechanical strength and sensitivity to fibrinolysis. Previous studies by Undas and co-workers [9,10] demonstrated that patients with stent thrombosis, compared to control PCI patients, formed denser fibrin clots which were more resistant to fibrinolysis. In these studies, 90% of the patients received a bare metal stent (BMS). However, the use of drug eluting stents (DES) compared to BMS has increased in recent years, which might have changed the risk factors associated with stent thrombosis. Therefore, we assessed the potential of plasma fibrin clot formation (lag time, maximal absorbance) and fibrinolysis (clot lysis time) from patients with a history of stent thrombosis compared to matched control patients in a currently representative cohort.
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2. Patients and methods 2.1. Study design and population A single-center case-control study including PCI patients with stent implantation was performed. Cases underwent an index PCI (PCI of initial stent implantation) after which they suffered from a definite stent thrombosis according to the Academic Research Consortium criteria [11]. The timing of stent thrombosis was divided into cases with early (≤ 30 days after index PCI) and late stent thrombosis (b 30 days after index PCI). Included cases had suffered from stent thrombosis between January 2007 and September 2011. Controls underwent an index PCI without suffering from stent thrombosis between index PCI and blood sampling. Control patients were matched 1:1 based on the indication (stable angina pectoris, unstable angina pectoris/non ST-segment elevation myocardial infarction, ST-segment elevation myocardial infarction) and time (± 14 days) of the index PCI from the cases. Subjects using oral anticoagulants or heparins at the time of blood collection were excluded. We selected all patients with stent thrombosis, who were matched with a control patient, and with available blood samples from the matched stent thrombosis and control patients. Written informed consent was provided by all participants. The study was approved by the local institutional Ethics Committee and was conducted according to the principles of the Declaration of Helsinki. 2.2. Blood collection and preparation All subjects had visited the St Antonius Hospital for blood sampling (Nieuwegein, The Netherlands). The minimal time interval between the last performed PCI and blood sampling was one month for cases and controls. Venous blood samples were collected from the antecubital vein using 21-gauge needles and Vacuette® tubes (Greiner Bio-one, Frickenhausen, Germany) containing 3.2% (w/v) sodium citrate. To avoid haemostatic activation, the first 5 ml of free-flowing blood was discarded. Platelet poor plasma was obtained by two separate centrifugation steps. Samples were first centrifuged for 10 min at 150g, followed by 15 min at 2500g. All platelet poor plasma samples were stored at −80 °C until analysis. 2.3. Fibrin clot formation and fibrinolysis assay To monitor fibrin clot formation, plasma samples were diluted 1.67 times with Hepes-buffer (25 mM Hepes (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid), 150 mM NaCl, pH = 7.5) and 125 μl of diluted plasma was transferred into a low binding polystyrene 96-well plate (Greiner, Frickenhausen, Germany). Fibrin polymerization was started by addition of 25 μl activation mixture. The activation mixture contained human thrombin (Enzyme Research Laboratories, Swansea, UK; final concentration: 0.75 nmol/l), phospholipids which were prepared by sonication as described earlier [12] (1,2-dioleoyl-sn-glycero3-phosphoserine, 1,2-dioeoyl-sn-glycero-3-phosphocholine, 1,2dioleoyl-snglycero-3-phosphoethanolamine (DOPS/DOPC/DOPE, 20/ 60/20, mol/mol/mol), Avanti Polar lipids Inc., Alabaster, Alabama, USA; final concentration: 10 μmol/l), and CaCl2 (final concentration: 16 mmol/l). Measurements were at 405 nm every 15 s for 60 min at 37 °C using an ELx808 plate reader (Biotek Instruments, Winooski, VT). The lag time, defined as the time to an increase of 0.01 optical density from baseline, together with the maximal absorbance were determined from the curves of the turbidity measurements. Turbidity measurements were performed in duplicate. To monitor fibrinolysis, recombinant tissue plasminogen activator (Boehringer Ingelheim, Alkmaar, the Netherlands; final concentration: 50 ng/ml) was added to the activation mixture, as described as above, at the start of the assay and turbidity was recorded for 6 h. Clot lysis
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time was calculated as the time from 50% clot formation to 50% fibrinolysis. Lysis measurements were performed in triplicate. The final concentrations of human thrombin and recombinant tissue plasminogen activator used in these assays were determined by turbidimetric assays with a normal plasma pool of healthy volunteers not using any medication. 2.4. Laboratory measurements Plasma concentrations of fibrinogen were measured using a Sysmex® CA-7000 System Automated Coagulation Analyzer with reagents obtained from Siemens Healthcare Diagnostics (Marburg, Germany) according to the Claus method [13]. Platelet count was measured using a LH 750 (Beckman Coulter) and cholesterol levels were using a Cobas 6000 (Roche Diagnostics). Hypercholesterolemia was defined as an increased level of total cholesterol (N 5.0 mmol/l), triglycerides (N 1.5 mmol/l), LDL (N 2.5 mmol/l) or a decreased level of HDL (b 1.0 mmol/l). 2.5. Statistical analysis Statistical analyses were performed with PRISM for Windows, version 5.00 (GraphPad Software, San Diego, CA, USA), and SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). Continuous data are expressed as mean ± standard deviation, and categorical data are expressed as frequencies no./total no. (%). Baseline characteristics and laboratory measurements at the time of blood sampling were analyzed using McNemar test or paired Student's t-test. Differences between cases and controls for fibrin clot properties were analyzed using univariate conditional logistic regression, reported as odds ratios (OR) with 95% confidence intervals (CI). Subgroup analysis was performed for cases with early vs. late stent thrombosis, and for cases with early stent thrombosis and their matched controls, as well as for cases with late stent thrombosis and their matched controls. A two-tailed p-value b 0.05 was considered as statistically significant. 3. Results 3.1. Patients A total of 27 cases and 27 controls were included. Twelve cases had experienced an early stent thrombosis and 15 cases a late stent thrombosis. None of the matched controls experienced a stent thrombosis between index PCI and blood sampling. Baseline characteristics are summarized in Table 1. Compared to controls, significantly more cases were current smokers at time of index PCI. The rate of hypertension was numerically higher for cases than for controls however without reaching statistical significance. The mean time-interval between stent thrombosis and blood sampling for cases was 35 ± 25 months and between index PCI and blood sampling for controls was 42 ± 26 months. More cases than controls were on dual antiplatelet therapy at the time of blood sampling (Table 2). Fibrinogen levels, cholesterol levels and platelet count were comparable between cases and matched controls. 3.2. Fibrin clot properties The fibrin clot properties were not significantly different between cases and controls (lag time: 173 ± 47 s vs. 162 ± 28 s, p = 0.18; maximal absorbance: 0.78 ± 0.16 vs. 0.83 ± 0.21, p = 0.36; clot lysis time: 69 ± 20 min vs. 71 ± 25 min, p = 0.78) (Fig. 1). Lag time, maximal absorbance, and clot lysis time were not associated with stent thrombosis (Table 3). Subgroup analyses for cases with early vs. late stent thrombosis showed no significant differences in fibrin clot properties (Table 4). Also, analyses of cases with early
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Fibrinogen levels were correlated with maximal absorbance for cases and controls (r = 0.91 and r = 0.91, both p b 0.001).
Table 1 Baseline characteristics at the time of index PCI.
Clinical characteristics Female Age (years) Body mass index (kg/m2) Current smoking Diabetes mellitus Hypertension Hypercholesterolemia Family history of CVD Medical history Prior MI Prior PCI Prior CABG Malignancy Renal failure PAD AF Thrombo-embolic events LVEF b 45% Indicationa Stable angina pectoris UAP/NSTEMI STEMI Procedural characteristics Bare metal stents Drug eluting stents Target vessel RCA LAD RCX Total stent length (mm) Antiplatelet therapy GP IIb/IIIa Aspirin Clopidogrel
Cases (n = 27) n (%)
Matched controls (n = 27) n (%)
p-Value
4 (14.8) 57 ± 11 26.3 ± 3.2 18 (72.0) 6 (22.2) 13 (48.1) 15 (60.0) 19 (73.1)
3 (11.1) 60 ± 9 26.9 ± 3.4 8 (32.0) 3 (11.1) 5 (18.5) 12 (48.0) 14 (53.8)
1.000 0.240 0.494 0.002 0.453 0.077 0.581 0.267
9 (33.3) 7 (25.9) 1 (3.7) 1 (3.7) 1 (3.8) 3 (11.1) 0 (0.0) 3 (11.1) 6 (37.5)
5 (18.5) 8 (29.6) 1 (3.7) 3 (11.1) 1 (3.8) 0 (0.0) 1 (3.7) 1 (3.7) 4 (25.0)
0.344 1.000 1.000 0.625 1.000 0.250 1.000 0.625 0.625
9 (33.3) 4 (14.8) 14 (51.9)
9 (33.3) 4 (14.8) 14 (51.9)
1.000 1.000 1.000
9 (33.3) 18 (66.7)
11 (40.7) 16 (59.3)
0.754 0.754
9/2 (33.3) 16 (59.3) 2 (7.4) 27.0 ± 13.6
11 (40.7) 9 (33.3) 7 (25.9) 23.3 ± 10.5
0.791 0.065 0.180 0.268
8 (29.6) 24 (96.0) 23 (92.0)
6 (22.2) 24 (96.0) 22 (88.0)
0.754 1.000 1.000
4. Discussion
PCI: percutaneous coronary intervention; BMI: body mass index; CVD: cardiovascular disease; MI: myocardial infarction; CABG: coronary artery bypass graft; PAD: peripheral artery disease; AF: atrial fibrillation; LVEF: left ventricular ejection fraction; UAP: unstable angina pectoris; NSTEMI: non-ST-segment elevation myocardial infarction; STEMI: STsegment elevation myocardial infarction; RCA: right coronary artery; LAD: left anterior descending artery; RCX: right circumflex artery; GP: glycoprotein. Continuous data are presented as mean ± standard deviation. a Matched item.
stent thrombosis vs. their matched controls and cases with late stent thrombosis vs. their matched controls showed no significant differences in fibrin clot properties (Table 5).
Table 2 Characteristics at the time of blood sampling.
Medication Aspirin DAPT Clopidogrel Prasugrel Ticagrelor Statins Laboratory measurements Fibrinogen (g/l) Platelet count (103/μl) Total cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Triglycerides (mmol/l)
Cases (n = 27) n (%)
Matched controls (n = 27) n (%)
p-Value
27 (100.0) 20 (74.1) 13 (48.1) 5 (18.5) 2 (7.4) 27 (100)
27 (100.0) 10 (37.0) 8 (29.6) 2 (7.4) 0 (0.0) 25 (92.6)
NA 0.01 0.27 0.38 0.50 0.50
3.6 ± 0.7 225 ± 42 4.1 ± 0.8 2.1 ± 0.6 1.28 ± 0.83 1.8 ± 0.7
3.6 ± 0.7 211 ± 51 4.1 ± 0.8 1.9 ± 0.6 1.20 ± 0.33 2.1 ± 1.3
0.88 0.34 0.97 0.12 0.71 0.33
NA: not applicable; DAPT: dual antiplatelet therapy; LDL: low density lipoprotein; HDL: high density lipoprotein. Continuous data are presented as mean ± standard deviation.
This study did not observe differences between in vitro fibrin clot formation and fibrinolysis in patients with a history of stent thrombosis and matched control PCI patients, suggesting that fibrin clot properties are not altered in patients with a history of stent thrombosis. Moreover, in the pathophysiology of stent thrombosis, plasma components play no important role in fibrin clot formation and fibrinolytic susceptibility. Our results are contrary to earlier observations of in vitro experiments [9,10]. Undas et al. showed altered fibrin clot properties and an impaired susceptibility to fibrinolysis for stent thrombosis patients compared to control PCI patients [9]. Our study methods differ from this study at three points. First, the methods of fibrin clot formation and fibrinolysis were slightly different. The concentration of thrombin for the turbidimetric assays was lower in our study, with a more sensitive assay as a result. Second, we matched control patients based on the indication and time of the index PCI of the case, whereas in the study of Undas et al. control patients were matched for numerous parameters, which could not always be realized. Case-control matching based on age and sex might be a more common method, nevertheless, we are convinced that matching based on indication for index PCI is more important, because the pathophysiology and/or clinical outcome of ST-segment elevation myocardial infarction and e.g. stable angina pectoris is different, and will be more suitable to discover predictors for stent thrombosis. Matching based on index PCI resulted in an equal follow-up period and a PCI procedure around the same period of time regarding stent placement and balloon dilatation methods. Our matching method still resulted in an equal distribution for age and sex. Third, the ratio of patients included with early vs. late stent thrombosis was different, respectively 12 (44%) vs. 15 (56%) for our study and 41 (87%) vs. 6 (13%) for the study by Undas. Literature described an impaired fibrinolytic potential for patients with early stent thrombosis compared to control PCI patients [10]. It could be hypothesized that the formation of a denser fibrin clot and hypofibrinolysis might be more important for the pathophysiology of early stent thrombosis than for late stent thrombosis, as the underlying pathophysiological mechanisms for early and late stent thrombosis also show differences. For example, delayed vascular healing is predominantly associated with late stent thrombosis [14], while discontinuation of clopidogrel is especially an important risk factor for the occurrence of early stent thrombosis [8]. Our subgroup analysis did not reveal a difference in fibrin clot formation and fibrinolysis between cases with early or late stent thrombosis, however no firm conclusions could be drawn from these analyses as the number of patients in the subgroups was small. One of the main determinants of fibrin clot structure is the concentration of thrombin [15]. The amount of thrombin in our in vitro experiments was kept constant and we cannot exclude that there might be a risk in vivo in the stented area for increased thrombin formation, which in case of clot formation will contribute to a denser fibrin clot structure. At the time of index PCI, more cases than controls were current smokers and tended to have more often hypertension. Smoking and hypertension both have an effect on fibrin clot properties. Smoking increases the clot strength and the resistance to fibrinolysis [16,17]. The use of antihypertensive agents increases the permeability and reduces the resistance to fibrinolysis in patients with arterial hypertension [18]. Whether smoking behavior was persistent until blood sampling is unknown and cannot be correlated to the fibrin clot properties in this study. At the time of blood sampling, more cases than controls were on dual antiplatelet therapy, as dual antiplatelet therapy was continued
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Fig. 1. Fibrin clot formation and fibrinolysis of cases and controls. (A) lag time, (B) maximal absorbance (A405 nm), and (C) clot lysis time in stent thrombosis patients (cases, n = 27) and matched control patients (controls, n = 27). Whiskers display mean ± standard deviation.
Table 3 Unadjusted OR of fibrin clot properties for stent thrombosis.
Lag time (s) Maximal absorbance CLT (min)
OR (95% CI)
p value
1.01 (0.99–1.03) 0.24 (0.01–4.97) 1.00 (0.98–1.02)
0.21 0.36 0.77
5. Conclusions
OR: odds ratio; CI: confidence interval; CLT: clot lysis time. Data are expressed as mean ± standard deviation.
Table 4 Fibrin clot properties of cases with early vs. late stent thrombosis. Early ST (n = 12)
Late ST (n = 15)
OR (95% CI)
measurements of individual fibrinolytic components. The causes of stent thrombosis were not included, which can be variable and are often unknown.
In conclusion, no differences were observed in fibrin clot formation and fibrinolysis in plasma of patients with a history of stent thrombosis compared with matched control patients, suggesting that fibrin clot formation and fibrinolysis do not have a significant role in the pathophysiology of stent thrombosis.
p-Value
Conflicts of interest
Lag time (s) 168 ± 49 178 ± 46 1.01 (0.99–1.02) 0.59 Maximal absorbance 0.74 ± 0.16 0.82 ± 0.16 33.83 (0.18–6420.35) 0.19 CLT (min) 62 ± 14 75 ± 23 1.05 (0.99–1.11) 0.14
None.
ST: stent thrombosis; OR: odds ratio; CI: confidence interval; CLT: clot lysis time. Data are expressed as mean ± standard deviation.
beyond one year after PCI in patients with stent thrombosis. Studies showed contrary results in clopidogrel altering fibrinogen levels [19, 20], another main determinant of fibrin clot structure [15]. However, fibrinogen levels were not significantly different between cases and controls in our study. Also, cholesterol levels and statin and aspirin use, factors that are known for their influence on fibrin clot formation and lysis [21], were not significantly different between cases and controls. Our study is hampered by the relatively small sample size and the small amount of platelet poor plasma, which limited the laboratory
Contributors TCG and JK designed and conducted the study, analyzed and interpreted the data and wrote the manuscript. JWG, JMB, CMH and HC designed the study, interpreted the data, and critically reviewed the manuscript.
Acknowledgements JK was funded by the Netherlands Heart Foundation (grant 2008B120). TCG was funded by the FP7 program of the European Commission (grant 260309). The funding sources were not involved in the establishment of the study and writing of the article.
Table 5 Fibrin clot properties of cases with early or late stent thrombosis and their matched controls. Cases with early stent thrombosis vs. controls
Lag time (s) Maximal absorbance CLT (min)
Cases (n = 12)
Matched controls (n = 12)
OR (95% CI)
p-Value
168 ± 49 0.74 ± 0.16 62 ± 14
158 ± 25 0.84 ± 0.18 75 ± 31
1.01 (0.98–1.03) 0.06 (0.00–7.11) 0.97 (0.93–1.02)
0.51 0.25 0.27
Cases (n = 15)
Matched controls (n = 15)
OR (95% CI)
p-Value
178 ± 46 0.82 ± 0.16 75 ± 23
164 ± 30 0.83 ± 0.24 68 ± 20
1.02 (0.99–1.05) 0.75 (0.01–40.13) 1.01 (0.98–1.05)
0.26 0.89 0.43
Cases with late stent thrombosis vs. controls
Lag time (s) Maximal absorbance CLT (min)
OR: odds ratio; CI: confidence interval; CLT: clot lysis time. Data are expressed as mean ± standard deviation.
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