Altered Plasma Fibrin Clot Properties Are Associated With In-Stent Thrombosis
Objectives— We sought to investigate whether patients with in-stent thrombosis (IST) display altered plasma fibrin clot properties.
Methods and Results— We studied 47 definite IST patients, including 15 with acute, 26 subacute and 6 late IST, and 48 controls matched for demographics, cardiovascular risk factors, concomitant treatment and angiographic/stent parameters. Plasma clot permeability (Ks), which indicates a pore size, turbidity (lag phase, indicating the rate of fibrin clot formation, ΔAbsmax, maximum absorbance of a fibrin gel, reflecting the fiber thickness), lysis time (t50%) and maximum rate of d-dimer release from clots (D-Drate) were determined 2 to 73 (median 14.7) months after IST. Patients with IST had 21% lower Ks, 14% higher ΔAbsmax, 11% lower D-Drate, 30% longer t50% (all P<0.0001) and 5% shorter lag phase compared to controls (P=0.042). There were no correlations between clot variables and the time of IST or that from IST to blood sampling. Multiple regression analysis showed that Ks (odds ratio=0.36 per 0.1 μm2, P<0.001), D-Drate (odds ratio=0.16 per 0.01 mg/L/min, P<0.001) and stent length (odds ratio=1.1 per 1 mm, P=0.043) were independent predictors of IST (R2=0.58, P<0.001).
Conclusions— IST patients tend to form dense fibrin clots resistant to lysis, and altered plasma fibrin clot features might contribute to the occurrence of IST.
There are several factors associated with increased risk of in-stent thrombosis (IST) in epicardial arteries, including the procedure itself (small final lumen dimension due to stent malapposition and/or underexpansion, stent length, placement of multiple stents, dissections), patient (low left ventricular ejection fraction [LVEF], diabetes mellitus, advanced age, stenting in acute coronary syndrome) and lesion characteristics (bifurcation, in-stent restenosis), stent design and premature cessation of antiplatelet drugs.1–3 Autopsy studies provided evidence that lack of complete endothelialization and persistent fibrin thrombi are a primary substrate underlying IST.4,5
Thrombin-mediated fibrinogen conversion to fibrin and fibrin monomer cross-linking result in the formation of a fibrin clot. Its structure and function are modulated by several genetic and environmental factors, predominantly those affecting levels and function of fibrinogen.6 Effective fibrinolysis is essential for removal of intravascular clots that might otherwise be manifest as thrombosis or enhance the development of atherosclerotic plaques. A more tightly packed and less porous fibrin structure reduces transport of proteins involved in fibrinolysis and anticoagulant reactions mediated by antithrombin inside the clot. These alterations might prolong the presence of fibrin in the lumen.6 Dense clot fiber networks with reduced susceptibility to lysis have been demonstrated in survivors of myocardial infarction (MI),7 in the acute phase of MI,8 and in patients with a history of the no-reflow phenomenon, defined as the absence of a complete myocardial perfusion despite successful opening of the infarct-related artery.9
Several lines of evidence10,11 indicate that incompletely endothelialized coronary stents predispose to fibrin deposition and persistent thrombi on the stent struts. However, it is not known whether the IST is associated with unfavorably altered fibrin clot properties that might contribute to this complication regardless of platelet reactivity. Therefore, we sought to evaluate plasma fibrin clot structure/function in patients with definite IST.
Ninety-five patients (76 men and 19 women), aged 60.9±9.9 years, who underwent percutaneous coronary intervention (PCI), were enrolled in the case-control study (Figure 1). We studied 47 patients with definite IST following initial PCI (the IST group). They were retrospectively identified based on hospital records and coronary angiography data performed in 6 catheterization laboratories in Poland in the years 2002 to 2008. All patients suspected of procedure-related IST or premature cessation of antiplatelet drugs were excluded. Forty-eight patients with no symptoms suggestive of IST, matched for age, sex, cardiovascular risk factors (previous MI, diabetes mellitus, dyslipidemia, smoking, hypertension, family history, renal failure), indications for initial PCI, angiographic parameters (culprit vessel, reference diameter of culprit vessel, TIMI (Thrombolysis in Myocardial Infarction) flow after PCI, the frequency of dissection not covered by stent), stent design (type, length) and concomitant treatment served as controls. They were selected from 15 473 patients who underwent coronary stenting at the same time as the IST patients. Definitions of IST proposed by Academic Research Consortium were used.12 According to the time elapsed since stent implantation, IST is classified as acute—during PCI or within the following 24 hours and subacute—between 1 to 30 days after PCI. The patients with late IST—between 30 days and 1 year after PCI or very late IST—more than 1 year after PCI were pooled, termed the late IST and analyzed collectively. The exclusion criteria were any acute illness, cancer, previous coronary artery bypass surgery, history of venous thromboembolism or stroke and anticoagulant therapy. All patients were followed and sampled at the outpatient clinics. The study was approved by the Ethics Committee of the Jagiellonian University. All subjects gave informed consent.
Coronary angiography performed at the time of stent implantation and during IST-related procedure was evaluated in patients with IST, whereas in controls a single angiography during stent implantation was analyzed. All coronary angiograms were analyzed off-line by 2 cardiologists (J.Z., M.K.) unaware of the fibrin clot data. They independently analyzed the reference lumen diameter of culprit vessel using Quantcor QCA software (Siemens, Germany). All coronary segments were carefully analyzed for the presence of dissection not covered by stent. Coronary stenosis was defined as significant based on visual inspection or when the degree of stenosis measured with QCA was >50%. Epicardial blood flow in culprit vessel was evaluated by means of the TIMI scale (from 0 to 3 with 3 indicating normal flow)13 and corrected TIMI frame count.14 Thrombus burden was evaluated using the TIMI Thrombus Grade.15
At the time of IST, we determined serum maximum levels of myocardial necrosis markers: that is, cardiac troponin I (TnIMAX) and creatine kinase-MB (CKMBMAX). Left ventricular ejection fraction was assessed by transthoracic echocardiography.
Follow-up data of patients treated at the outpatient clinic were recorded. Cardiac function was assessed using the New York Heart Association (NYHA) functional scale.
Blood was drawn from an antecubital vein with minimal stasis. Lipid profiles, blood cell counts, platelets, glucose, creatinine and d-dimer were assayed by routine laboratory techniques. Plasma samples (9:1 of 3.2% sodium citrate) were centrifuged within 20 minutes of collection and stored at −80°C. Fibrinogen was determined using the von Clauss method. High-sensitivity C-reactive protein was measured by latex nephelometry (Dade Behring). To assess fibrinolytic parameters, we measured tissue-type plasminogen activator activity, plasminogen activator inhibitor-1 activity (Biopool), and plasmin-antiplasmin complexes complexes (DRG Diagnostica). Plasma markers of platelet activation, including soluble CD40 ligand (sCD40L; R&D Systems), P-selectin (R&D Systems), β-thromboglobulin (Diagnostica Stago), and serum thromboxane B2, an indicator of aspirin’s action in vivo (Cayman Chemicals) were measured.
Permeation of plasma fibrin clots was investigated as previously described.8,16 Briefly, 20 mmol/L calcium chloride and 1 U/mL human thrombin (Sigma) were added to citrated plasma. Tubes containing the clots were connected to a reservoir of a TBS buffer (0.01 mol/L Tris, 0.1 mol/L NaCl, pH 7.5), and its volume flowing through the gels was measured within 60 minutes. A permeation coefficient (Ks), which indicates the pore size, was calculated from the equation: Ks=QxLxη/txAxΔp, where Q is the flow rate in time t, L is the length of a fibrin gel, η is the viscosity of liquid (in poise), A is the cross-sectional area (in cm2), and Δp is a differential pressure (in dyne/cm2). The interassay variability was 7.4%.
Citrated plasma was mixed (3:2) with 0.7 IU/mL thrombin, 0.1% Tween 80 and 20 mmol/L calcium chloride in TBS, and then clots were formed in tubes prepared like in the permeation experiments.17 After centrifugation at 6000g for 60 seconds, the volume of the supernatant evacuated from the tubes was assessed on the basis of the difference in weight of the tube. Compaction was expressed as this volume divided by the initial plasma volume used to form the fibrin clot. The interassay variability was 6.9%.
Plasma samples were diluted 1:1 with a buffer (0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, pH 7.4) and with addition of 1 U/mL human thrombin (Sigma) and 15 mmol/L calcium chloride to plasma-initiated polymerization.8,16 Absorbance was read at 405 nm for 15 minutes with a Perkin-Elmer Lambda 4B spectrophotometer (Molecular Devices Corp). The lag phase of the turbidity curve, which is the time required for fibrin protofibrils to grow to sufficient length to allow lateral aggregation to occur and this parameter is sensitive to among others fibrinopeptide A cleavage, and maximum absorbance at plateau (ΔAbsmax), which reflects the number of protofibrils per fiber, were recorded.16 The interassay and intraassay coefficients of variation were <7%.
Clot Lysis Assays
Assay 1. Fibrinolysis in the presence of recombinant tissue plasminogen activator (Boerhinger Ingelheim) was evaluated as previously described.16,18 Briefly, 100-μL citrated plasma was diluted with 100 μL of the same buffer containing 20-mmol/L calcium chloride, 1 U/mL human thrombin (Sigma) and 14-μmol/L recombinant tissue plasminogen activator. Assembly kinetics were monitored by absorbance at 405 nm for 30 minutes in duplicates. The time required for a 50% decrease in clot turbidity (t50%) from a peak value was chosen as a marker of the clot susceptibility to fibrinolysis. The interassay coefficient of variation was 6.2%.
Assay 2. Fibrin clots formed as described above were perfused with the same buffer containing 0.2 μmol/L recombinant tissue plasminogen activator.16,19 The lysis rate was determined by measuring the concentration of d-dimers (American Diagnostica), a marker of plasmin-mediated fibrin degradation, every 20 minutes in the effluent. Maximum rates of increase in d-dimer levels (D-Drate, mg/L/min), and maximum concentrations (D-Dmax) detected at 80 or 100 minutes were analyzed in each subject. The experiment was stopped when the fibrin gel collapsed. The interassay coefficients of variation was <7%.
The study was powered to have an 90% chance of detecting a 15% difference in plasma clot permeability and lysis time using a P value of 0.05, based on the values of fibrin features in the published article.9 In order to demonstrate such a difference or greater, 23 patients were required in each group. For a P value of 0.001, 45 patients per group were required.
Statistical analyses were performed with SPSS 12.01 software. Continuous variables are expressed as mean±SD or median (interquartile range) and categorical variables as number (percentage). Continuous variables were first checked for normal distribution by the Shapiro-Wilk statistic and the Bera-Jarque test and compared by Student t test when normally distributed or by the Mann-Whitney U test for nonnormally distributed variables. ANOVA was used to compare the differences among fibrin clot properties in relation to the time of IST. Categorical variables were compared by χ2 test. The Pearson or Spearman rank correlation coefficients were calculated to test the association between 2 variables with a normal or nonnormal distribution, respectively. All clinical and angiographic variables that showed the association with IST in univariate model (P≤0.2) and did not show substantial correlations (r>0.5) with another independent variable were then included in the stepwise multiple logistic regression analysis. P<0.05 was considered statistically significant.
The acute, subacute, and late IST was diagnosed in 15 (32%), 26 (55.3%) and 6 (12.7%) patients, respectively. The IST patients and controls did not differ with regard to age, sex, classic risk factors, fibrinogen, high-sensitivity C-reactive protein, lipid profile, fibrinolytic or platelet markers, indications to initial PCI and stent implantation and concomitant treatment during initial PCI (Table 1). There were no intergroup differences with regard to the indication to the first PCI, culprit vessel, a type of stent (Table 1) and the time from the first PCI and blood sampling. Patients with previous IST and the controls had similar baseline LVEF (50 [44 to 60] versus 55 [42 to 60] %, respectively), reference vessel diameter (3.04±0.36 versus 3.09±0.44 mm), stent length (20.3±7.6 versus 18.2±4.7 mm), TIMI flow before (1.62±1.34 versus 1.14±1.30) and after PCI (2.94±0.25 versus 2.98±0.14), maximal pressure of stent implantation (14.2±2.3 versus 13.9±3.1 atm), the frequency of bifurcation stenting (6.4 versus 2.1%) and dissections not covered by stent during the initial PCI (4.3 versus 2.1%). The IST patients had higher platelet count (Table 1) and higher thrombus burden after the first PCI (0.17±0.48 versus 0.02±0.14, P=0.046). Patients with acute, subacute, and late IST did not differ with regard to age, laboratory and angiographic variables (Supplemental Table I, available at http://atvb.ahajournals.org).
At the time of IST, balloon angioplasty was performed in 27 patients and an additional stent was implanted in 20 patients. Aspiration thrombectomy was used in 14 patients. During PCI for IST TIMI flow was improved from 0.64±1.15 to 2.77±0.56. Distal post-PCI embolization was observed in 11 patients.
Clot Permeability and Compaction
Plasma fibrin clots from the IST patients were significantly less permeable and showed less compaction than clots from controls without IST event (Table 2). The lowest permeability was found in the IST patients in whom STEMI (ST-Elevation Myocardial Infarction) was an indication for initial PCI. They had significantly lower Ks than control subjects with STEMI as the initial diagnosis (5.7±1.0 versus 7.5±1.0 10−9 cm2, P<0.001). Ks was also lower in patients with occlusive IST (TIMI-0/1 before PCI) compared to the remaining IST patients (5.7±1.2 versus 6.8±1.3 10−9 cm2, P=0.017). There were no differences in Ks and compaction with regard to culprit artery or cardiovascular risk factors (data not shown). This also held true for the time from stent implantation to IST (Supplemental Figure I, available at http://atvb.ahajournals.org). Permeability showed no association with the time from stent implantation or IST to the day of blood sampling (r<0.15, P>0.2). Ks was negatively correlated with fibrinogen levels (r=−0.51, P<0.001) and corrected TIMI frame count after IST-related PCI (r=−0.54, P<0.001). There were no correlations between Ks or compaction and age, routine laboratory tests, fibrinolytic markers, reference lumen diameter of culprit vessel, TnIMAX, CK-MBMAX, and LVEF (data not shown).
Compared with controls, IST patients had shorter lag phase and greater maximum clot absorbancy (ΔAbsmax; Table 2). Lag phase and ΔAbsmax were not associated with age, reference lumen diameter of culprit vessel, corrected TIMI frame count and LVEF. There were no differences in lag phase and ΔAbsmax with regard to culprit vessel and initial diagnosis (data not shown) as well as the time from stent implantation to IST (Supplemental Figure I, available at http://atvb.ahajournals.org). Turbidity variables showed no associations with the time from stent implantation or IST to blood collection (r≤0.1. P>0.2).
In the IST group, t50% was significantly longer compared with controls. Slower d-dimer release from clots and increased maximum d-dimer levels were also observed (Table 2). Interestingly, the IST patients in whom stent was implanted during STEMI had longer t50% (10.3±1.1 versus 9.0±1.3 minutes, P=0.006) and higher D-Dmax (4.4 [4.2 to 4.85] versus 3.5 [3.35 to 4.6] mg/L, P<0.05) compared with controls with stent implanted during STEMI. D-Drate, D-Dmax, and t50% did not differ with regard to culprit artery, cardiovascular risk factors and the type of IST (data not shown). Out of fibrinolytic markers only plasminogen activator inhibitor-1 correlated with D-Drate in IST patients (r=0.52, P=0.002), but not in controls. Lysis parameters were not associated with the time since stent implantation or IST to blood sampling (|r|<0.15, P>0.2). In IST patients, both t50% and D-Dmax were correlated with fibrinogen (r=0.31, P=0.003, and r=0.5, P<0.001, respectively) and corrected TIMI frame count (r=0.36, P=0.015, and r=0.34, P=0.024, respectively). D-Drate, D-Dmax and t50% showed no associations with glucose, platelet count, high-sensitivity C-reactive protein, reference lumen diameter of culprit vessel, CK-MBMAX, after IST-related PCI, and LVEF (data not shown).
Before IST event all patients received aspirin and 43 (91.5%) subjects took thienopyridines. At the time of IST all patients received a loading dose of 300 to 600 mg clopidogrel before PCI and 34 (72.3%) of patients were treated with abciximab. At blood sampling all patients took aspirin (75 to 150 mg/d), whereas 16 controls and 24 IST patients received clopidogrel. A dose of aspirin and clopidogrel treatment did not influence fibrin properties in either group (data not shown).
Independent Predictors of In-Stent Thrombosis
The stepwise logistic regression analysis for IST as a dependent variable is shown in Figure 2. Before the inclusion to the multiple analysis model, all univariate associations were analyzed (Supplemental Table II, available at http://atvb.ahajournals.org). There were significant correlations between the plasma fibrin variables (r=−0.64 for Ks and t50%, r=−0.64 for Ks and D-Dmax, r=0.54 for Ks and compaction, r=0.6 for t50% and D-Dmax, r=0.54 for t50% and ΔAbsmax, r=0.53 for ΔAbsmax and lag phase, P<0.001 for all). The multivariate model showed that D-Drate, Ks and stent length were the only independent predictors of IST (R2=0.58, P<0.001; Figure 2).
This study demonstrates that altered plasma clot properties and impaired susceptibility to lysis are associated with a history of definite IST. In patients with IST, plasma fibrin clots are formed more rapidly and have a more compact structure compared to those made from plasma obtained from patients who did not experience such a complication. Importantly, plasma clots of IST patients are more resistant to lysis, likely because of less efficient transport of fibrinolytic agents through a dense fibrin network, which configuration has been reported to have a stronger effect on the fibrinolysis rate than fiber thickness.20 Their findings suggest that altered plasma clot properties may characterize patients with previous IST and might help identify subjects at increased risk of such an event. Our results support autopsy and angioscopic findings10,11 by showing that certain unfavorable clot features might predispose to IST through the formation of compact thrombi resistant to lysis.
There is a clear relationship between the time course of IST and the degree of endothelialization after stent placement. It has been demonstrated that the time required to establish complete endothelialization after bare metal stent implantation is approximately 1 to 3 months.21 Angioscopy showed that 3 to 6 months after PCI subclinical thrombus deposition was seen in 33% to 89% of drug-eluting stents and 14% to 29% of bare metal stent-treated patients.11 Our findings provide a potential explanation for these observations showing that among coronary artery disease patients who typically display altered plasma fibrin clot features, those with extremely low clot permeability, low susceptibility to lysis, and large clot mass of rapid formation are particularly prone to develop IST after coronary stent implantation. It might be speculated that patients with “better” clot properties are at lower risk of developing IST despite the fact that they may have some thrombotic material on the surface of incompletely endothelialized coronary stents.
Determinants of Fibrin Clot Variables
Mechanisms by which IST is linked to altered plasma fibrin clot structure/function remain obscure. The IST patients and controls did not differ with regard to fibrinogen levels, a major predictor of fibrin clot properties.6 In patients with or without a history of IST, there were similar high-sensitivity C-reactive protein levels, which when elevated reduce clot permeability and fibrinolysis.16 No associations were observed between fibrin features and platelet or fibrinolytic markers. Moreover, alterations in fibrin function/structure, found in patients a few months after IST, cannot be attributed to activation of coagulation observed shortly after acute coronary ischemia. Additional potential mechanism behind the reported differences in plasma clot features might be oxidative stress that has been shown to alter fibrin properties when cardiovascular risk is high.8 It has been demonstrated that genetic factors contribute modestly to variance in fibrin clot measures, including lysis time (range, 10% to 40%), whereas the contribution of environmental factors, being poorly characterized, is much larger.22 It is likely that environmental factors modulating fibrin(ogen) function can account for IST-related differences in plasma fibrin clot properties.
We failed to show any differences in fibrin clot properties in patients with acute, subacute and late IST, suggesting that certain clot features are common to all patients with previous IST regardless of the time of the event. If so, it remains to be established which other factors might explain differences in the time of the IST event in the so-defined “IST-prone” population of coronary artery disease patients. Most of our IST cases occurred within 30 days after PCI, and given the number of patients the findings for this type of IST appear the most reliable. A limited number of patients with late IST hampers drawing conclusive results on late IST.
Importantly, altered plasma clot properties in the IST patients were apparent despite administration of aspirin that can increase clot permeability and promote fibrin lysis.23 Because all patients were taking aspirin, the impact of aspirin could not have been assessed. Beneficial effects of statins on clot properties have also been described.16,24 In the current study, IST-related differences were observed regardless of statin use or not. No data are available on any fibrin-modulating effects of clopidogrel in plasma-based assays; however, thromboelastography of whole blood samples indicates varying changes in clot properties associated with clopidogrel administration.25
In search for factors associated with increased risk of IST, we have demonstrated that an increase in pore size by every 0.1 μm2 or d-dimer release acceleration of every 0.01 mg/L/min are associated with reduction of the IST risk by 2.8 and 6.3 times, respectively. Our findings indicate that clot permeability and velocity of fibrin lysis are the best predictors of IST and might be used in clinical studies.
First, the number of the patients enrolled was small. However, the IST patients were meticulously matched with controls; therefore, the intergroup differences in clot properties reported here are most likely associated with the IST occurrence alone. Second, our analysis was based on a determination of each variable at a single time point after IST. Third, blood samples taken before IST or stent implantation were not available. However, samples taken during an acute event display markedly, but transiently, altered clot features.8 Fourth, noncompliance to clopidogrel or aspirin, together with potential resistance to these agents, at the time of IST have not been specifically addressed in this study. Analysis of material obtained during thrombectomy in acute clinical setting of IST might provide additional information. However, detailed quantitative analysis of such thrombi is not well established. Homocysteine and lipoprotein(a), which can alter fibrin properties,26 have not been determined in this study. However, there is no evidence showing increased levels of both variables in IST patients. Contrary to most data, largely determined in purified systems, indicating that decreased fibrin clot permeability is associated with thinner fibers and lower clot turbidity, we observed increased maximum absorbancy. It seems that in plasma-based assays reduced permeability is frequently associated with higher absorbancy.8,27 Finally, our experimental approach did not allow the assessment of the effect of blood cells and platelets on fibrin clot structure/function, which can reduce the rate of fibrinolysis.28 Given the higher platelet count in the IST group, this suggests that plasma fibrin parameters may underestimate IST-related alterations in thrombi.
Our findings suggest that altered plasma fibrin clot properties associated with attenuated fibrinolysis can be detected in patients with IST. Reduced clot permeability and susceptibility to lysis represent a novel prothrombotic mechanism, which might contribute to the IST. Prospective studies in stable patients undergoing coronary stenting are needed to determine clinical implications of our intriguing observations.
Sources of Funding
This work was supported by a grant of Jagiellonian University (to A.U.).
A.U. and J.Z. equally contributed to this work.
Received July 24, 2009; revision accepted October 22, 2009.
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