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Stent implantation in the superficial femoral artery: Short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis
Thrombosis Research 130 (2012) 485–490
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Regular Article
Stent implantation in the superficial femoral artery: Short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis Gerhard Cvirn a,⁎, Gerd Hoerl a, Axel Schlagenhauf b, Erwin Tafeit a, Marianne Brodmann c, Guenther Juergens a, Martin Koestenberger b, Thomas Gary c a b c
Institute of Physiological Chemistry, Medical University of Graz, Austria Department of Pediatrics, Medical University of Graz, Austria Division of Angiology, Medical University of Graz, Austria
a r t i c l e
i n f o
Article history: Received 25 January 2012 Received in revised form 23 March 2012 Accepted 9 April 2012 Available online 29 April 2012 Keywords: Coagulation In-stent restenosis Superficial femoral artery Thrombelastometry Thrombin generation
a b s t r a c t Introduction: The mechanisms of restenosis, the recurrence of luminal narrowing, are complex and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, presumably contributes to the formation of in-stent restenosis (ISR). It was therefore the aim of our study to investigate whether blood coagulation/ thrombin generation plays a critical role in the formation of ISR in peripheral artery disease patients with stent angioplasty in the superficial femoral artery. Materials and Methods: We aimed to examine in this retrospective study whether patients with high-degree restenosis (50-75% lumen diameter reduction, n = 20) are in a hypercoaguable state implying enhanced readiness to generate thrombin compared to patients with low-degree restenosis (b 50% lumen diameter reduction, n = 14). Results: The coagulation tests calibrated automated thrombography, activated partial thromboplastin time, platelet aggregation, platelet adhesion, fibrinogen, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two patient groups. However, the thrombelastometry-derived value Coagulation Time (CT) was significantly shorter in the high-degree restenosis group (p= 0.012), indicating a hypercoagulable state of patients with high-degree restenosis. Under our experimental conditions, CTs shorter than 444.5 s identify patients at high risk (sensitivity = 95%) for luminal narrowing. Conclusions: Our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development of ISR in peripheral arteries after stent insertion and that the thrombelastometry-derived CT might be a suitable value to identify peripheral artery disease patients at risk for development of high-degree instent restenosis in the superficial femoral artery. © 2012 Elsevier Ltd. All rights reserved.
Introduction Treatment of superficial femoral artery (SFA) disease by stent implantation has demonstrated superiority to percutaneous transluminal angioplasty (PTA) alone. However, in-stent restenosis (ISR) remains a frequent complication [1]. The mechanisms of restenosis are complex Abbreviations: APTT, activated partial thromboplastin time; ETP, endogenous thrombin potential; CAT, calibrated automated thrombography; CFT, clot formation time; CRP, C-reactive protein; CT, coagulation time; F 1 + 2, prothrombin fragment 1 + 2; GPRP, fibrin polymerization inhibitor H-Gly-Pro-Arg-Pro-OH; HDL, high-density lipoprotein; ISR, in-stent restenosis; LDL, low-density lipoprotein; MCF, maximum clot firmness; PAD, peripheral arterial disease; PCI, percutaneous coronary intervention; PPP, platelet poor plasma; PT, prothrombin time; PTA, percutaneous transluminal angioplasty; PTCA, percutaneous transluminal coronary angioplasty; SD, standard deviation; SFA, superficial femoral artery; TEM, thrombelastometry; TF, lipidated tissue factor; WB, whole blood. ⁎ Corresponding author at: Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse 21/II, A-8010 Graz, Austria. Tel.: + 43 316 380 4174; fax: + 43 316 380 9610. E-mail address: [email protected] (G. Cvirn). 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.04.007
and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, has recently been recognized as an important factor in the development of restenosis [2–5]. Thrombin supports restenosis via activation of thrombin receptors on endothelial cells, smooth muscle cells, macrophages, and fibroblasts [6,7]. Thus, a critical role of blood coagulation implying thrombin generation has to be expected in the development of ISR in the SFA in patients with peripheral arterial disease (PAD). However, Wahlgren et al. did not observe an influence of thrombin generation on stent restenosis in thirty-four patients with PAD undergoing angioplasty of the iliac and superficial femoral arteries [8]. In this study, plasma levels of prothrombin fragment 1 + 2 (F 1 + 2) served as an indicator of thrombin generation. There were no significant changes in the plasma levels of F 1 + 2 after PTA. Moreover, F 1 + 2 level was no statistically significant predictor of luminal narrowing or restenosis. The aim of our study was therefore to further scrutinize whether development of ISR after stent implantation in the SFA in patients with PAD is promoted by a hypercoagulable state and enhanced
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thrombin generation, respectively. For the present study, thirty-four patients were admitted to the department of angiology of the university hospital Graz/Austria approximately three years after SFA-stent insertion. Twenty out of thirty-four patients presented with highdegree restenosis, defined as 50–75 % lumen diameter reduction at the site of PTA. We examined whether patients with high-degree ISR are hypercoagulable/generate higher amounts of thrombin when compared to patients with low-degree ISR. Patients’ coagulable state was assessed particularly by measuring thrombin generation curves and by monitoring whole blood clot development courses. Thrombin generation curves were detected in platelet poor plasma (PPP) samples by means of calibrated automated thrombography (CAT) [9,10]. Whole blood clot development courses were monitored by means of tissue factor (TF) triggered thrombelastometry (TEM). This method has been shown to allow a sensitive and a close to the in-vivo situation estimation of the patients’ coagulable state [11]. In order to extend the haemostatic profiling in our patients with low vs. high-degree restenosis, we additionally determined activated partial thromboplastin times (APTTs) as well as platelet adhesion and aggregation. Moreover, a possible platelet-activating effect of the implanted stent might result in shedding of procoagulant microparticles. Therefore, we also determined microparticles’ procoagulant activities in both patient groups.
levels of fibrinogen were determined on a Schnitger-Gros coagulometer according to the Clauss-method. Whole-blood aggregation experiments were performed on the Chrono-Log Whole Blood Aggregometer Model 590 from Probe & Go (Endingen, Germany). Platelet adhesion was measured on a Cone and Platelet Analyzer (CPA) from DiaMed (Linz, Austria). Microparticles’ procoagulant activity was determined by the functional assay ZYMUPHEN MP-Activity from HYPHEN BioMed (Neuville, France). Microplate Scanning Spectrometer was purchased from Bio-Tek Instruments, Inc., Winooski, Vermont, USA. Collection of WB and Preparation of Plasma
Materials and Methods
Venous blood samples were obtained from 34 PAD patients with SFA-stent implantation in. The study was approved by the appropriate Institutional Review Board. Informed consent was obtained. Blood (2.7 ml) was collected into precitrated S-Monovette premarked tubes (three from each individual) from Sarstedt (Nümbrecht, Germany), containing 300 μl of 0.106 mol/l sodium citrate. The first tube aspirated was discarded. The whole blood from the two remaining tubes was pooled, and subsequently used for determination of blood cell counts as well as for thrombelastometry and platelet function measurements. The remaining whole blood was centrifuged at room temperature for 15 min at 1200 ×g to obtain PPP for subsequent determination of standard coagulation times, fibrinogen plasma concentrations, thrombin generation curves, and microparticles’ procoagulant activity.
Patients
Automated Fluorogenic Measurement of the Thrombin Generation
The diagnosis of PAD was assigned in our outpatient clinic by means of clinical evaluation, ankle-brachial index, and duplex scan. The current study is based on 34 patients scheduled for stent implantation throughout the years 2007 and 2008. The results presented herein are derived from blood collected from the same patients between February and April 2011. Stent implantation was applied as secondary stent implantation for unsatisfactory results of percutaneous transluminal angioplasty (PTA) alone, which means residual stenosis of >50%, flowlimiting dissection, elastic recoil, or acute thrombotic occlusion in the area of balloon angioplasty. A standardized protocol was used for stent implantation. All our patients were treated with self-expandable nitinol stents (Absolute stent from Abbot Vascular, Des Plaines, IL). During the intervention, 3000 IU of unfractionated heparin was administered to avoid stent occlusion. After the procedure, patients were treated with low-molecular-weight heparin (enoxaparin 40 mg twice daily) for 48 h. All patients received antithrombotic medication with 100 mg aspirin before intervention and 100 mg aspirin and 75 mg clopidogrel after stent implantation for 3 month. After 3 month, the patients were treated with 100 mg aspirin indefinitely. Our patients were divided into two groups: patients with low-degree restenosis (b50% lumen diameter reduction, n = 14) and patients with highdegree restenosis (50-75% lumen diameter reduction, n = 20). ISR was assessed with duplex scan. An obstruction was considered as hemodynamically relevant when the grade of stenosis was above 50%, defined as at least doubling of the peak velocity in the obstruction area. The exact degree of stenosis was calculated by means of an area stenosis degree in the cross-sectional view.
Measurement of the thrombin generation was performed using CAT [9]. The ability of a given plasma sample to generate thrombin was assessed with respect to lag time preceding the thrombin burst (Lag Time), time to peak (ttPeak), peak height (Peak), and endogenous thrombin potential (ETP), and the time point at which free thrombin has disappeared (StartTail). Measurements were carried out in the presence of 5 pmol/l of tissue factor (TF) (final concentration).
Reagents and Devices Blood cell counts were determined on a Sysmex KX-21 N Automated Hematology Analyzer from Sysmex (Illinois, USA). Thrombin generation curves were monitored by means of CAT purchased from Thrombinoscope BV, Maastricht, the Netherlands. The TEM coagulation analyser (ROTEM®05) was purchased from Matel Medizintechnik, Graz, Austria. Activated partial thromboplastin time and prothrombin time were measured on the optomechanical coagulation analyzer Behring Fibrintimer from Behring Diagnostics GmbH, Marburg, Germany. Plasma
WB Tissue Factor Triggered TEM Assay We obtained the following values: Coagulation Time (CT), the period of time from initiation of the test to the initial fibrin formation; Clot Formation Time (CFT), time of beginning of clot formation until the amplitude of thrombelastogram reaches 20 mm; Maximum Clot Firmness (MCF), expressing the maximum strength in millimeters of the final clot; and Alpha, the angle between the line in the middle of the TEM tracing and the line tangential to the developing “body” of the TEM tracing. The alpha angle represents the acceleration (kinetics) of fibrin build up and cross-linking. This method has been described in detail recently (11). Whole Blood Platelet Aggregation Assay WB aggregation was assayed with WB aggregometer by the impedance method [12,13]. Impedance aggregometry results are expressed as “amplitude (or maximum aggregation) [ohm]” at 6 minutes after reagent addition and as “lag time (or aggregation time) [seconds]”, the time interval until the onset of platelet aggregation. The rate of platelet aggregation is expressed as “slope [ohm/min]”. Collagen and endogenously generated thrombin were used as platelet agonists, respectively, described in our previous study [11]. Whole Blood Platelet Adhesion Assay The method has been described in detail previously [14]. Briefly, 130 μl of citrated WB were placed in polystyrene tubes and subjected to flow (1300 s − 1) for 2 minutes using a rotating Teflon cone. The wells were washed with phosphate buffered saline, stained with
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May-Grünwald solution and analyzed with an image analysis system. The two platelet function values Surface Coverage (SC) and Average Size (AS) were evaluated. SC is defined as the percentage of total area covered by platelets. AS is defined as the average size of the surface bound objects [15].
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in the SFA three years ago. Twenty out of these 34 patients presented with high-degree restenosis. No statistical significant differences in demographic data and blood cell counts were found between patients with low vs. high-degree ISR. We aimed to compare the procoagulatory state of patients with low-degree restenosis with that of patients with high-degree ISR.
Microparticles’ Procoagulant Activity Thrombin Generation in Patients with Low- vs. High- degree Restenosis Plasma samples, supplemented with calcium, FXa and FIIa inhibitors, were introduced to microplate wells coated with Streptavidine and biotinylated Annexin V. FXa-FVa mixtures containing calcium and purified prothrombin were introduced. Microparticles bind to Annexin V and expose their phospholipid surface, thus allowing conversation of prothrombin to thrombin. The phospholipid concentration is the limiting factor. The amount of thrombin generation, reflecting the phospholipid concentration, is measured via its specific activity on a chromogenic thrombin substrate. Absorbance was measured at 405 nm. Statistics Calculations were performed by SPSS 18.0 (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean ± SD. Differences between patients with low and high degree restenosis were analyzed by means of t-test for independent samples in case of normally distributed variables [16], otherwise the Mann–Whitney U-test was applied (17). A P-value less than 0.05 was considered as statistically significant. Furthermore, to investigate the discrimination power of our variables to distinguish between the two patient groups, stepwise discriminant analysis and ROC curve analysis were applied [17,18].
Thrombin generation was comparatively evaluated in the two patient groups applying CAT with respect to Lag Time, ttPeak, Peak, ETP, and Start Tail. No statistical significant differences were found between patients with low- vs. high-degree of restenosis (Table 2). Thrombelastometry in Patients with Low- vs. High-degree Restenosis Thrombelastometry values were comparatively evaluated in the two patient groups with respect to CT, CFT, MCF, and alpha angle. Coagulation Times and the sum of Coagulation Time and Clot formation Time were significantly shorter and alpha angle was significantly higher in patients with high-degree restenosis compared to those of patients with lowdegree restenosis (Table 2). No statistical significant differences concerning CFT and MCF were found between the two patient groups (Table 2). APTT in Patients with Low- vs. High-degree Restenosis No statistical significant differences were found between patients with low- (39.56±5.61 s) vs. high-degree of restenosis (38.44 ±3.57 s).
Results
Whole Blood Platelet Aggregation in Patients with Low- vs. High- degree Restenosis
Table 1 shows the patients’ characteristics. We included 34 consecutive patients with PAD undergoing secondary stent implantation
Whole blood platelet aggregation values were comparatively evaluated in the two patient groups with respect to Amplitude, Slope, and
Table 1 Patients’ characteristics. A total of 34 PAD patients with stent implantation three year ago were included in the study. Data presented are from follow-up procedure. No statistical significant differences were found between the groups.
Male, n (%) Age (years), mean ± SD Blood cell counts WBC (x 10³/μl), mean ± SD RBC (x 106/μl), mean ± SD Platelets (x 10³/μl), mean ± SD Clinical characteristics Current smoker, n (%) Hypertension, n (%) Diabetes, n (%) BMI, mean ± SD Coronary heart disease, n (%) Carotid artery stenosis, n (%) Cholesterol [mg/dl], mean ± SD CRP [mg/l], mean ± SD HDL [mg/dl], mean ± SD LDL [mg/dl], mean ± SD Concomitant therapy Statin therapy, n (%) Beta blocker therapy, n (%) PPI therapy, n (%) Position stent set in SFA, n (%) Position I (proximal) Position II (middle) Position III (distal) Time elapsed since stent implantation (month), mean ± SD Occlusion prior to intervention, n (%) Lesion length prior to Intervention (mm), mean ± SD Stent length (mm), mean ± SD
All patients (n = 34)
Patients with low degree restenosis (b50% lumen diameter reduction) (n= 14, Grp. 1)
Patients with high degree restenosis (50-75% lumen diameter reduction) (n= 20, Grp. 2)
P-value (Grp. 1 vs. Grp 2)
21 (61.8) 72.0 ± 10.9
9 (64.3) 73.7 ± 9.4
12 (60.0) 70.9 ± 11.9
0.604 0.469
6.6 ± 1.8 4.1 ± 0.5 213.2 ± 106.8
7.0 ± 2.1 4.3 ± 0.6 197.0 ± 54.0
6.3 ± 1.5 4.0 ± 0.4 224.7 ± 132.2
0.288 0.085 0.466
6 (17.6) 30 (88.2) 16 (47.1) 27.7 ± 3.3 10 (29.4) 19 (55.9) 179.4 ± 40.8 4. 8 ± 6.2 53.2 ± 16.9 98.2 ± 37.1
3 (21.4) 14 (100.0) 7 (50.0) 27.9 ± 3.6 5 (35.7) 7 (50.0) 172.4 ± 34. 5 5.0 ± 5.1 49.4 ± 12.0 92.7 ± 28.5
3 (15.0) 16 (80.0) 9 (45.0) 27.4 ± 3.2 5 (25.0) 12 (60.0) 184.4 ± 44.7 4.6 ± 7.1 55.9 ± 19.5 102.1 ± 42.4
0.571 0.091 0.632 0.673 0.515 0.905 0.407 0.877 0.283 0.476
25 (73.5) 19 (55.9) 16 (47.1)
12 (85.7) 9 (64.3) 5 (35.7)
13 (65.0) 10 (50.0) 11 (55.0)
0.189 0.289 0.281
1 (2.9) 11 (32.4) 23 (67.6) 36 ± 16.2 17 (50.0) 78 ± 57.0 68.9 ± 31.4
0 5 (35.7) 10 (71.4) 35 ± 18.7 7 (50.0) 80 ± 69.2 75.7 ± 37.4
1 (5.0) 6 (30.0) 13 (65.0) 37 ± 13.4 10 (50.0) 78.5 ± 49.7 64.5 ± 27.4
0.411 0.736 0.704 0.718 1.000 0.942 0.320
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Table 2 Thrombin generation values, thrombelastometry values, and whole blood aggregation values in patients with low vs. high degree restenosis. Data are presented as mean ± SD.
Thrombin generation values: Lag Time (min) ttPeak (min) Peak (nmol/l) ETP (nmol/L · min) Start Tail (min) Thrombelastometry values: Coagulation Time (s) Clot Formation Time (s) Maximum Clot Firmness (mm) Alpha (°) Sum: Coagulation Time + Clot Formation Time (s) Whole blood aggregation values: WB platelet aggregation provoked by collagen Amplitude (ohm) Slope (ohm/min) Lag Time (s) WB platelet aggregation provoked by endogenously generated thrombin Amplitude (ohm) Slope (ohm/min) Lag Time (s)
Patients with low degree restenosis (b 50% lumen diameter reduction) (n = 14, Grp. 1)
Patients with high degree restenosis (50-75% lumen diameter reduction) (n = 20, Grp. 2)
P-values (Grp. 1 vs. Grp. 2)
1.94 ± 0.29 3.54 ± 0.22 457.35 ± 69.20 1779.04 ± 306.15 17.43 ± 2.90
1.94 ± 0.28 3.55 ± 0.34 476.25 ± 53.67 1817.43 ± 268.41 16.98 ± 2.40
0.959 0.938 0.376 0.701 0.628
430.89 ± 72.05 186.68 ± 93.82 58.21 ± 7.28 57.75 ± 11.06 617.57 ± 159.22
371.75 ± 57.99 134.10 ± 30.61 61.15 ± 4.76 64.55 ± 4.75 505.85 ± 66.23
0.012 0.052 0.161 0.045 0.025
8.21 ± 3.12 4.79 ± 2.05 112.79 ± 35.32
7.50 ± 4.75 4.10 ± 2.65 132.60 ± 65.97
0.626 0.423 0.314
10.57 ± 3.39 6.50 ± 2.77 59.14 ± 27.00
10.06 ± 3.85 6.41 ± 2.94 51.18 ± 34.42
0.700 0.933 0.486
Lag Time. No statistical significant differences were found between the two patient groups (Table 2). Whole Blood Platelet Adhesion in Patients with Low- vs. High- Degree Restenosis Whole blood platelet adhesion values were comparatively evaluated in the two patient groups with respect to Surface Coverage and Average Size. No statistical significant differences were found between the two patient groups concerning Surface Coverage (9.87 ± 4.91 vs. 10.55 ± 3.66 %) and Average Size (34.96 ± 10.24 vs. 39.10 ± 17.18 μm²). Microparticles’ Procoagulant Activity in Patients with Low- vs. HighDegree Restenosis Microparticles’ procoagulant activity, evaluated by means of enzymelinked immunosorbent assay, was comparatively evaluated in the two patient groups. No statistical significant differences were found between patients with low- (24.04± 7.88 nmol/l) vs. high-degree (28.32 ± 12.59 nmol/l) restenosis. Three Standard Coagulation Values Measured Prior to Stent Implantation In order to investigate whether standard coagulation parameters could serve as predictors of stent narrowing and restenosis, we compared APTT, PT, and plasma fibrinogen concentrations in the two patient groups in pre-intervention plasma samples. No statistical significant differences between the groups were found concerning APTT (37.26 ± 9.39 vs. 34.78 ± 6.95 s), PT (98.71 ± 16.53 vs. 98.75 ± 24.39 s), and plasma fibrinogen concentration (427.29 ± 99.75 vs. 400.40 ± 141.23 mg/dl). Identification of Patients at Risk for High- Degree Restenosis Using TEM-derived CTs To calculate the discrimination power between the two patient groups stepwise discriminant analysis was applied on our data. Only TEM-derived CT was significantly selected (p = 0.012), providing a sensitivity of 75.0 % and a specificity of 57.1 %. Twenty three out of 34 patients (67.6 %) were correctly classified by this CT value.
Furthermore, ROC curve analysis was applied on significantly different variables. Again the best discrimination result was achieved by CT with an area index of 0.709, a sensitivity of95 %, a specificity of 50 %, and 76.5 % correctly classified cases (26 of 34) at an optional cutoff value for the two patient groups of 444.5 s. Thus, under our experimental conditions, CT values shorter than 444.5 s identify patients at high risk (sensitivity = 95 %) for luminal narrowing in stents implanted in the SFA.
Discussion Previous studies suggest that thrombin, the pivotal enzyme in haemostasis, plays a critical role in the formation of ISR [4,5]. Thrombin is a powerful stimulator for the migration of macrophages and for the proliferation of smooth muscle cells thereby contributing to the later stages of the myoproliferative process in the pathogenesis of restenosis [19–21]. For example, high plasma levels of Heparin Cofactor II, a physiological inhibitor of thrombin actions, have been shown to be associated with reduced incidence of ISR after percutaneous coronary intervention [22]. Moreover, artificial thrombin inhibitors like recombinant hirudin or inogatran have been shown to reduce restenosis after angioplasty in animal models [23,24]. Therefore, in the present study, we investigated whether blood coagulation plays a role in the formation of ISR in PAD patients with stent angioplasty in the SFA. The coagulation status of 14 patients with low-degree restenosis was compared with that of 20 patients with high-degree restenosis three years after stent placement. The coagulation tests CAT, APTT, platelet aggregation, platelet adhesion, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two groups. Even levels of fibrinogen, a strong biochemical predictor of restenosis after coronary angioplasty [25–27], were comparable in both groups before, and, as indicated by same TEM-derived MCF values, three years after stent placement. However, the two TEM-derived values Coagulation Time (CT) and alpha angle were significantly deranged in the direction of hypercoagulability in the high-degree restenosis group. Whereas the alpha angle, representing the kinetics of fibrin build up and cross-linking, received only borderline significance (p = 0.045), CTs were markedly shorter in the high- degree restenosis group (p = 0.012).
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Sorensen et al. have shown that short CTs indicate fast thrombin generation [28]. Thus, a hypercoagulable state of patients with highdegree restenosis compared to patients with low-degree restenosis was detected in the present study by means of TEM employing activation with minute amounts of TF. To our knowledge, no studies exist to date reporting on the influence of blood coagulation/thrombin generation on the process of restenosis in PAD patients with stent implantation in the SFA. However, Schoebel et al. investigated the relevance of haemostasis on restenosis in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). They found that a hypercoagulable disease state prior to intervention characterizes a high-risk group prone for restenosis in clinically stable coronary artery disease [29]. In a similar study Salvioni et al. have shown a relation between thrombin activation soon after PTCA and late restenosis [30]. Gurbel et al. investigated the relation of ex vivo platelet reactivity, fibrin generation, and thrombin-induced clot strength to postdischarge ischemic events in patients undergoing percutaneous coronary intervention (PCI) over six month [1]. They found significantly higher platelet reactivity and fibrin formation, and, even more predictive, higher clot strength evaluated by means of TEM in patients with ischemic events after PCI. Thus, similar to our present study, a TEM derived parameter apparently allows identification of patients at high risk for thromboembolic events. In accordance, Wilson et al. have shown a significant correlation between hypercoagulability as determined by means of TEM and the development of deep venous thrombosis in 250 patients with femoral neck fracture [31]. In addition to these studies we show herein that patients with low-degree ISR are in a hypercoagulable state compared to patients with low-degree ISR not only before and immediately after, as shown by Schoebel et al. and Salvioni et al., but also three years after stent placement. In our previous study we have shown that patients with high risk for developing ISR and occlusion have elevated Apo B levels and low HDL cholesterol [32]. We assumed that platelet CD 36 interacts with oxidized lipoproteins, resulting in enhanced platelet reactivity [33]. However, the results in the present study do not support this assumption. Under our experimental conditions, platelet function was essentially the same in the low- and the high-degree ISR groups. The results from our present study suggest that the TEM-derived CT might be a suitable diagnostic parameter to reveal patients with a hypercoagulable state associated with elevated risk for formation of luminal narrowing and of ISR. However, CTs in our study were determined three years after stent placement. Thus, a further (prospective) study is needed to clarify whether TEM-derived CTs could actually serve as a predictor for ISR. This is in accordance with the call of Dai et al. for more prospective studies to clarify the predictive accuracy of TEM for postoperative thromboembolic events[34]. In this future study, TEM-derived CTs should be determined immediately before and at several time points after stent placement. It should be investigated whether short pre-angioplasty CTs correlate with enhanced formation of ISR. A limitation of our study is the small sample size as we evaluated only one vessel segment to minimize possible bias [35,36]. On the other hand, our patients are homogenous for the vessel segment treated and for the kind of endovascular treatment, which could be considered as the strength of our study. Further limitations of our study are that coagulability was not assessed during the time of neointimal formation and that platelet reactivity was determined when patients were off of clopidogrel. Neointimal formation is the major cause of ISR and high on-clopidogrel treatment is a major risk factor for recurrent ischemic events including target revascularization for ISR. Moreover, we did not perform arachidonic acid-induced platelet aggregation measurements in order to detect a possible influence of patients’ aspirin intake on platelet aggregation parameters. In conclusion, our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development
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of ISR and that the TEM-derived CT might be a suitable value to identify PAD patients at risk for formation of high-degree ISR in the SFA. Conflict of Interest Statement All authors have made substantial contributions to the conception and design of the study, to the acquisition of data or to the analysis and interpretation of data. All authors declare no conflict of interest. Acknowledgement We are indebted to E. Mathew and H. Moussalli for assistance with the laboratory assays. References [1] Gurbel PA, Bliden KP, Guyer K, Cho PW, Zaman KA, Bassi AK, et al. Platelet reactivity in patients and recurrent events post-stenting: results of the PREPARE POST-STENTING Study. J Am Coll Cardiol 2005;46:1820–6. [2] Aihara K, Azuma H, Akaike M, Kurobe H, Takamori N, Ikeda Y, et al. Heparin cofactor II is an independent protective factor against peripheral arterial disease in elderly subjects with cardiovascular risk factors. J Atheroscler Thromb 2009;16: 127–34. [3] Hering J, Amann B, Angelkort B, Rottmann M. Thrombin-antithrombin complex and the prothrombin fragment in arterial and venous blood of patients with peripheral arterial disease. Vasa 2003;32:193–7. [4] Hasenstab D, Lea H, Hart CE, Lok S, Clowes AW. Tissue factor overexpression in rat arterial neointima models thrombosis and progression of advanced atherosclerosis. Circulation 2000;101:2651–7. [5] Patterson C, Stouffer GA, Madamanchi N, Runge MS. New tricks for old dogs: nonthrombotic effects of thrombin in vessel wall biology. Circ Res 2001;88:987–97. [6] Siller-Matula JM, Haberl K, Prillinger K, Panzer S, Lang I, Jilma B. The effect of antiplatelet drugs clopidogrel and aspirin is less immediately after stent implantation. Thromb Res 2009;123:874–80. [7] Wilensky RL, Pyles JM, Fineberg N. Increased thrombin activity correlates with increased ischemic event rate after percutaneous transluminal coronary angioplasty: lack of efficacy of locally delivered urokinase. Am Heart J 1999;138 [319–5]. [8] Wahlgren CM, Sten-Linder M, Egberg N, Kalin B, Blohme L, Swedenborg J. The role of coagulation and inflammation after angioplasty in patients with peripheral arterial disease. Cardiovasc Intervent Radiol 2006;29:530–5. [9] Hemker HC, Giesen P, Al Dieri R, Regnault V, De Smedt E, Wagenvoord R, et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33:4–15. [10] Butenas S, Van't Veer C, Mann KG. ‘Normal’ thrombin generation. Blood 1999;94: 2169–78. [11] Cvirn G, Gallistl S, Kutschera J, Wagner T, Ferstl U, Jurgens G, et al. Collagen/endogenous thrombin-induced platelet aggregation in whole blood samples. Blood Coagul Fibrinolysis 2007;18:585–8. [12] Nylander S, Johansson K, Van Giezen JJ, Lindahl TL. Evaluation of platelet function, a method comparison. Platelets 2006;17:49–55. [13] Sweeney JD, Hoernig LA, Michnik A, Fitzpatrick JE. Whole blood aggregometry. Influence of sample collection and delay in study performance on test results. Am J Clin Pathol 1989;92:676–9. [14] Varon D, Darkik R, Shenkman B, Kotev-Emeth S, Farzame N, Tamarin I, et al. A new method for quantitative analysis of whole blood platelet interaction with extracellular matrix under flow conditions. Thromb Res 1997;85:283–94. [15] Linder N, Shenkman B, Levin E, Sirota L, Vishne TH, Tamarin I, et al. Deposition of whole blood platelets on extracellular matrix under flow conditions in preterm infants. Arch Dis Child Fetal Neonatal Ed 2002;86:F127–30. [16] Tafeit E, Moller R, Sudi K, Reibnegger G. The determination of three subcutaneous adipose tissue compartments in non-insulin-dependent diabetes mellitus women with artificial neural networks and factor analysis. Artif Intell Med 1999;17: 181–93. [17] Tafeit E, Moller R, Jurimae T, Sudi K, Wallner SJ. Subcutaneous adipose tissue topography (SAT-Top) development in children and young adults. Coll Antropol 2007;31:395–402. [18] Tafeit E, Moller R, Sudi K, Reibnegger G. ROC and CART analysis of subcutaneous adipose tissue topography (SAT-Top) in type-2 diabetic women and healthy females. Am J Hum Biol 2000;12:388–94. [19] Graham DJ, Alexander J. The effects of thrombin on bovine aortic endothelial and smooth muscle cells. J Vasc Surg 1990;11:307–11. [20] Mc Namara CA, Sarembock IJ, Gimple LW, Fenton JW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest 1993;91:94–8. [21] Walters TK, Gorog DA, Wood RFM. Thrombin generation following arterial injury is a critical event in the pathogenesis of the proliferative stages of the atherosclerotic process. J Vasc Res 1994;31:173–7. [22] Takamori N, Azuma H, Kato M, Hashizume S, Aihara K, Akaike M, et al. High plasma heparin cofactor II activity is associated with reduced incidence of in-stent restenosis after percutaneous coronary intervention. Circulation 2004;109:481–6.
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Regular Article
Stent implantation in the superficial femoral artery: Short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis Gerhard Cvirn a,⁎, Gerd Hoerl a, Axel Schlagenhauf b, Erwin Tafeit a, Marianne Brodmann c, Guenther Juergens a, Martin Koestenberger b, Thomas Gary c a b c
Institute of Physiological Chemistry, Medical University of Graz, Austria Department of Pediatrics, Medical University of Graz, Austria Division of Angiology, Medical University of Graz, Austria
a r t i c l e
i n f o
Article history: Received 25 January 2012 Received in revised form 23 March 2012 Accepted 9 April 2012 Available online 29 April 2012 Keywords: Coagulation In-stent restenosis Superficial femoral artery Thrombelastometry Thrombin generation
a b s t r a c t Introduction: The mechanisms of restenosis, the recurrence of luminal narrowing, are complex and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, presumably contributes to the formation of in-stent restenosis (ISR). It was therefore the aim of our study to investigate whether blood coagulation/ thrombin generation plays a critical role in the formation of ISR in peripheral artery disease patients with stent angioplasty in the superficial femoral artery. Materials and Methods: We aimed to examine in this retrospective study whether patients with high-degree restenosis (50-75% lumen diameter reduction, n = 20) are in a hypercoaguable state implying enhanced readiness to generate thrombin compared to patients with low-degree restenosis (b 50% lumen diameter reduction, n = 14). Results: The coagulation tests calibrated automated thrombography, activated partial thromboplastin time, platelet aggregation, platelet adhesion, fibrinogen, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two patient groups. However, the thrombelastometry-derived value Coagulation Time (CT) was significantly shorter in the high-degree restenosis group (p= 0.012), indicating a hypercoagulable state of patients with high-degree restenosis. Under our experimental conditions, CTs shorter than 444.5 s identify patients at high risk (sensitivity = 95%) for luminal narrowing. Conclusions: Our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development of ISR in peripheral arteries after stent insertion and that the thrombelastometry-derived CT might be a suitable value to identify peripheral artery disease patients at risk for development of high-degree instent restenosis in the superficial femoral artery. © 2012 Elsevier Ltd. All rights reserved.
Introduction Treatment of superficial femoral artery (SFA) disease by stent implantation has demonstrated superiority to percutaneous transluminal angioplasty (PTA) alone. However, in-stent restenosis (ISR) remains a frequent complication [1]. The mechanisms of restenosis are complex Abbreviations: APTT, activated partial thromboplastin time; ETP, endogenous thrombin potential; CAT, calibrated automated thrombography; CFT, clot formation time; CRP, C-reactive protein; CT, coagulation time; F 1 + 2, prothrombin fragment 1 + 2; GPRP, fibrin polymerization inhibitor H-Gly-Pro-Arg-Pro-OH; HDL, high-density lipoprotein; ISR, in-stent restenosis; LDL, low-density lipoprotein; MCF, maximum clot firmness; PAD, peripheral arterial disease; PCI, percutaneous coronary intervention; PPP, platelet poor plasma; PT, prothrombin time; PTA, percutaneous transluminal angioplasty; PTCA, percutaneous transluminal coronary angioplasty; SD, standard deviation; SFA, superficial femoral artery; TEM, thrombelastometry; TF, lipidated tissue factor; WB, whole blood. ⁎ Corresponding author at: Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse 21/II, A-8010 Graz, Austria. Tel.: + 43 316 380 4174; fax: + 43 316 380 9610. E-mail address: [email protected] (G. Cvirn). 0049-3848/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.04.007
and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, has recently been recognized as an important factor in the development of restenosis [2–5]. Thrombin supports restenosis via activation of thrombin receptors on endothelial cells, smooth muscle cells, macrophages, and fibroblasts [6,7]. Thus, a critical role of blood coagulation implying thrombin generation has to be expected in the development of ISR in the SFA in patients with peripheral arterial disease (PAD). However, Wahlgren et al. did not observe an influence of thrombin generation on stent restenosis in thirty-four patients with PAD undergoing angioplasty of the iliac and superficial femoral arteries [8]. In this study, plasma levels of prothrombin fragment 1 + 2 (F 1 + 2) served as an indicator of thrombin generation. There were no significant changes in the plasma levels of F 1 + 2 after PTA. Moreover, F 1 + 2 level was no statistically significant predictor of luminal narrowing or restenosis. The aim of our study was therefore to further scrutinize whether development of ISR after stent implantation in the SFA in patients with PAD is promoted by a hypercoagulable state and enhanced
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thrombin generation, respectively. For the present study, thirty-four patients were admitted to the department of angiology of the university hospital Graz/Austria approximately three years after SFA-stent insertion. Twenty out of thirty-four patients presented with highdegree restenosis, defined as 50–75 % lumen diameter reduction at the site of PTA. We examined whether patients with high-degree ISR are hypercoagulable/generate higher amounts of thrombin when compared to patients with low-degree ISR. Patients’ coagulable state was assessed particularly by measuring thrombin generation curves and by monitoring whole blood clot development courses. Thrombin generation curves were detected in platelet poor plasma (PPP) samples by means of calibrated automated thrombography (CAT) [9,10]. Whole blood clot development courses were monitored by means of tissue factor (TF) triggered thrombelastometry (TEM). This method has been shown to allow a sensitive and a close to the in-vivo situation estimation of the patients’ coagulable state [11]. In order to extend the haemostatic profiling in our patients with low vs. high-degree restenosis, we additionally determined activated partial thromboplastin times (APTTs) as well as platelet adhesion and aggregation. Moreover, a possible platelet-activating effect of the implanted stent might result in shedding of procoagulant microparticles. Therefore, we also determined microparticles’ procoagulant activities in both patient groups.
levels of fibrinogen were determined on a Schnitger-Gros coagulometer according to the Clauss-method. Whole-blood aggregation experiments were performed on the Chrono-Log Whole Blood Aggregometer Model 590 from Probe & Go (Endingen, Germany). Platelet adhesion was measured on a Cone and Platelet Analyzer (CPA) from DiaMed (Linz, Austria). Microparticles’ procoagulant activity was determined by the functional assay ZYMUPHEN MP-Activity from HYPHEN BioMed (Neuville, France). Microplate Scanning Spectrometer was purchased from Bio-Tek Instruments, Inc., Winooski, Vermont, USA. Collection of WB and Preparation of Plasma
Materials and Methods
Venous blood samples were obtained from 34 PAD patients with SFA-stent implantation in. The study was approved by the appropriate Institutional Review Board. Informed consent was obtained. Blood (2.7 ml) was collected into precitrated S-Monovette premarked tubes (three from each individual) from Sarstedt (Nümbrecht, Germany), containing 300 μl of 0.106 mol/l sodium citrate. The first tube aspirated was discarded. The whole blood from the two remaining tubes was pooled, and subsequently used for determination of blood cell counts as well as for thrombelastometry and platelet function measurements. The remaining whole blood was centrifuged at room temperature for 15 min at 1200 ×g to obtain PPP for subsequent determination of standard coagulation times, fibrinogen plasma concentrations, thrombin generation curves, and microparticles’ procoagulant activity.
Patients
Automated Fluorogenic Measurement of the Thrombin Generation
The diagnosis of PAD was assigned in our outpatient clinic by means of clinical evaluation, ankle-brachial index, and duplex scan. The current study is based on 34 patients scheduled for stent implantation throughout the years 2007 and 2008. The results presented herein are derived from blood collected from the same patients between February and April 2011. Stent implantation was applied as secondary stent implantation for unsatisfactory results of percutaneous transluminal angioplasty (PTA) alone, which means residual stenosis of >50%, flowlimiting dissection, elastic recoil, or acute thrombotic occlusion in the area of balloon angioplasty. A standardized protocol was used for stent implantation. All our patients were treated with self-expandable nitinol stents (Absolute stent from Abbot Vascular, Des Plaines, IL). During the intervention, 3000 IU of unfractionated heparin was administered to avoid stent occlusion. After the procedure, patients were treated with low-molecular-weight heparin (enoxaparin 40 mg twice daily) for 48 h. All patients received antithrombotic medication with 100 mg aspirin before intervention and 100 mg aspirin and 75 mg clopidogrel after stent implantation for 3 month. After 3 month, the patients were treated with 100 mg aspirin indefinitely. Our patients were divided into two groups: patients with low-degree restenosis (b50% lumen diameter reduction, n = 14) and patients with highdegree restenosis (50-75% lumen diameter reduction, n = 20). ISR was assessed with duplex scan. An obstruction was considered as hemodynamically relevant when the grade of stenosis was above 50%, defined as at least doubling of the peak velocity in the obstruction area. The exact degree of stenosis was calculated by means of an area stenosis degree in the cross-sectional view.
Measurement of the thrombin generation was performed using CAT [9]. The ability of a given plasma sample to generate thrombin was assessed with respect to lag time preceding the thrombin burst (Lag Time), time to peak (ttPeak), peak height (Peak), and endogenous thrombin potential (ETP), and the time point at which free thrombin has disappeared (StartTail). Measurements were carried out in the presence of 5 pmol/l of tissue factor (TF) (final concentration).
Reagents and Devices Blood cell counts were determined on a Sysmex KX-21 N Automated Hematology Analyzer from Sysmex (Illinois, USA). Thrombin generation curves were monitored by means of CAT purchased from Thrombinoscope BV, Maastricht, the Netherlands. The TEM coagulation analyser (ROTEM®05) was purchased from Matel Medizintechnik, Graz, Austria. Activated partial thromboplastin time and prothrombin time were measured on the optomechanical coagulation analyzer Behring Fibrintimer from Behring Diagnostics GmbH, Marburg, Germany. Plasma
WB Tissue Factor Triggered TEM Assay We obtained the following values: Coagulation Time (CT), the period of time from initiation of the test to the initial fibrin formation; Clot Formation Time (CFT), time of beginning of clot formation until the amplitude of thrombelastogram reaches 20 mm; Maximum Clot Firmness (MCF), expressing the maximum strength in millimeters of the final clot; and Alpha, the angle between the line in the middle of the TEM tracing and the line tangential to the developing “body” of the TEM tracing. The alpha angle represents the acceleration (kinetics) of fibrin build up and cross-linking. This method has been described in detail recently (11). Whole Blood Platelet Aggregation Assay WB aggregation was assayed with WB aggregometer by the impedance method [12,13]. Impedance aggregometry results are expressed as “amplitude (or maximum aggregation) [ohm]” at 6 minutes after reagent addition and as “lag time (or aggregation time) [seconds]”, the time interval until the onset of platelet aggregation. The rate of platelet aggregation is expressed as “slope [ohm/min]”. Collagen and endogenously generated thrombin were used as platelet agonists, respectively, described in our previous study [11]. Whole Blood Platelet Adhesion Assay The method has been described in detail previously [14]. Briefly, 130 μl of citrated WB were placed in polystyrene tubes and subjected to flow (1300 s − 1) for 2 minutes using a rotating Teflon cone. The wells were washed with phosphate buffered saline, stained with
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May-Grünwald solution and analyzed with an image analysis system. The two platelet function values Surface Coverage (SC) and Average Size (AS) were evaluated. SC is defined as the percentage of total area covered by platelets. AS is defined as the average size of the surface bound objects [15].
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in the SFA three years ago. Twenty out of these 34 patients presented with high-degree restenosis. No statistical significant differences in demographic data and blood cell counts were found between patients with low vs. high-degree ISR. We aimed to compare the procoagulatory state of patients with low-degree restenosis with that of patients with high-degree ISR.
Microparticles’ Procoagulant Activity Thrombin Generation in Patients with Low- vs. High- degree Restenosis Plasma samples, supplemented with calcium, FXa and FIIa inhibitors, were introduced to microplate wells coated with Streptavidine and biotinylated Annexin V. FXa-FVa mixtures containing calcium and purified prothrombin were introduced. Microparticles bind to Annexin V and expose their phospholipid surface, thus allowing conversation of prothrombin to thrombin. The phospholipid concentration is the limiting factor. The amount of thrombin generation, reflecting the phospholipid concentration, is measured via its specific activity on a chromogenic thrombin substrate. Absorbance was measured at 405 nm. Statistics Calculations were performed by SPSS 18.0 (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean ± SD. Differences between patients with low and high degree restenosis were analyzed by means of t-test for independent samples in case of normally distributed variables [16], otherwise the Mann–Whitney U-test was applied (17). A P-value less than 0.05 was considered as statistically significant. Furthermore, to investigate the discrimination power of our variables to distinguish between the two patient groups, stepwise discriminant analysis and ROC curve analysis were applied [17,18].
Thrombin generation was comparatively evaluated in the two patient groups applying CAT with respect to Lag Time, ttPeak, Peak, ETP, and Start Tail. No statistical significant differences were found between patients with low- vs. high-degree of restenosis (Table 2). Thrombelastometry in Patients with Low- vs. High-degree Restenosis Thrombelastometry values were comparatively evaluated in the two patient groups with respect to CT, CFT, MCF, and alpha angle. Coagulation Times and the sum of Coagulation Time and Clot formation Time were significantly shorter and alpha angle was significantly higher in patients with high-degree restenosis compared to those of patients with lowdegree restenosis (Table 2). No statistical significant differences concerning CFT and MCF were found between the two patient groups (Table 2). APTT in Patients with Low- vs. High-degree Restenosis No statistical significant differences were found between patients with low- (39.56±5.61 s) vs. high-degree of restenosis (38.44 ±3.57 s).
Results
Whole Blood Platelet Aggregation in Patients with Low- vs. High- degree Restenosis
Table 1 shows the patients’ characteristics. We included 34 consecutive patients with PAD undergoing secondary stent implantation
Whole blood platelet aggregation values were comparatively evaluated in the two patient groups with respect to Amplitude, Slope, and
Table 1 Patients’ characteristics. A total of 34 PAD patients with stent implantation three year ago were included in the study. Data presented are from follow-up procedure. No statistical significant differences were found between the groups.
Male, n (%) Age (years), mean ± SD Blood cell counts WBC (x 10³/μl), mean ± SD RBC (x 106/μl), mean ± SD Platelets (x 10³/μl), mean ± SD Clinical characteristics Current smoker, n (%) Hypertension, n (%) Diabetes, n (%) BMI, mean ± SD Coronary heart disease, n (%) Carotid artery stenosis, n (%) Cholesterol [mg/dl], mean ± SD CRP [mg/l], mean ± SD HDL [mg/dl], mean ± SD LDL [mg/dl], mean ± SD Concomitant therapy Statin therapy, n (%) Beta blocker therapy, n (%) PPI therapy, n (%) Position stent set in SFA, n (%) Position I (proximal) Position II (middle) Position III (distal) Time elapsed since stent implantation (month), mean ± SD Occlusion prior to intervention, n (%) Lesion length prior to Intervention (mm), mean ± SD Stent length (mm), mean ± SD
All patients (n = 34)
Patients with low degree restenosis (b50% lumen diameter reduction) (n= 14, Grp. 1)
Patients with high degree restenosis (50-75% lumen diameter reduction) (n= 20, Grp. 2)
P-value (Grp. 1 vs. Grp 2)
21 (61.8) 72.0 ± 10.9
9 (64.3) 73.7 ± 9.4
12 (60.0) 70.9 ± 11.9
0.604 0.469
6.6 ± 1.8 4.1 ± 0.5 213.2 ± 106.8
7.0 ± 2.1 4.3 ± 0.6 197.0 ± 54.0
6.3 ± 1.5 4.0 ± 0.4 224.7 ± 132.2
0.288 0.085 0.466
6 (17.6) 30 (88.2) 16 (47.1) 27.7 ± 3.3 10 (29.4) 19 (55.9) 179.4 ± 40.8 4. 8 ± 6.2 53.2 ± 16.9 98.2 ± 37.1
3 (21.4) 14 (100.0) 7 (50.0) 27.9 ± 3.6 5 (35.7) 7 (50.0) 172.4 ± 34. 5 5.0 ± 5.1 49.4 ± 12.0 92.7 ± 28.5
3 (15.0) 16 (80.0) 9 (45.0) 27.4 ± 3.2 5 (25.0) 12 (60.0) 184.4 ± 44.7 4.6 ± 7.1 55.9 ± 19.5 102.1 ± 42.4
0.571 0.091 0.632 0.673 0.515 0.905 0.407 0.877 0.283 0.476
25 (73.5) 19 (55.9) 16 (47.1)
12 (85.7) 9 (64.3) 5 (35.7)
13 (65.0) 10 (50.0) 11 (55.0)
0.189 0.289 0.281
1 (2.9) 11 (32.4) 23 (67.6) 36 ± 16.2 17 (50.0) 78 ± 57.0 68.9 ± 31.4
0 5 (35.7) 10 (71.4) 35 ± 18.7 7 (50.0) 80 ± 69.2 75.7 ± 37.4
1 (5.0) 6 (30.0) 13 (65.0) 37 ± 13.4 10 (50.0) 78.5 ± 49.7 64.5 ± 27.4
0.411 0.736 0.704 0.718 1.000 0.942 0.320
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Table 2 Thrombin generation values, thrombelastometry values, and whole blood aggregation values in patients with low vs. high degree restenosis. Data are presented as mean ± SD.
Thrombin generation values: Lag Time (min) ttPeak (min) Peak (nmol/l) ETP (nmol/L · min) Start Tail (min) Thrombelastometry values: Coagulation Time (s) Clot Formation Time (s) Maximum Clot Firmness (mm) Alpha (°) Sum: Coagulation Time + Clot Formation Time (s) Whole blood aggregation values: WB platelet aggregation provoked by collagen Amplitude (ohm) Slope (ohm/min) Lag Time (s) WB platelet aggregation provoked by endogenously generated thrombin Amplitude (ohm) Slope (ohm/min) Lag Time (s)
Patients with low degree restenosis (b 50% lumen diameter reduction) (n = 14, Grp. 1)
Patients with high degree restenosis (50-75% lumen diameter reduction) (n = 20, Grp. 2)
P-values (Grp. 1 vs. Grp. 2)
1.94 ± 0.29 3.54 ± 0.22 457.35 ± 69.20 1779.04 ± 306.15 17.43 ± 2.90
1.94 ± 0.28 3.55 ± 0.34 476.25 ± 53.67 1817.43 ± 268.41 16.98 ± 2.40
0.959 0.938 0.376 0.701 0.628
430.89 ± 72.05 186.68 ± 93.82 58.21 ± 7.28 57.75 ± 11.06 617.57 ± 159.22
371.75 ± 57.99 134.10 ± 30.61 61.15 ± 4.76 64.55 ± 4.75 505.85 ± 66.23
0.012 0.052 0.161 0.045 0.025
8.21 ± 3.12 4.79 ± 2.05 112.79 ± 35.32
7.50 ± 4.75 4.10 ± 2.65 132.60 ± 65.97
0.626 0.423 0.314
10.57 ± 3.39 6.50 ± 2.77 59.14 ± 27.00
10.06 ± 3.85 6.41 ± 2.94 51.18 ± 34.42
0.700 0.933 0.486
Lag Time. No statistical significant differences were found between the two patient groups (Table 2). Whole Blood Platelet Adhesion in Patients with Low- vs. High- Degree Restenosis Whole blood platelet adhesion values were comparatively evaluated in the two patient groups with respect to Surface Coverage and Average Size. No statistical significant differences were found between the two patient groups concerning Surface Coverage (9.87 ± 4.91 vs. 10.55 ± 3.66 %) and Average Size (34.96 ± 10.24 vs. 39.10 ± 17.18 μm²). Microparticles’ Procoagulant Activity in Patients with Low- vs. HighDegree Restenosis Microparticles’ procoagulant activity, evaluated by means of enzymelinked immunosorbent assay, was comparatively evaluated in the two patient groups. No statistical significant differences were found between patients with low- (24.04± 7.88 nmol/l) vs. high-degree (28.32 ± 12.59 nmol/l) restenosis. Three Standard Coagulation Values Measured Prior to Stent Implantation In order to investigate whether standard coagulation parameters could serve as predictors of stent narrowing and restenosis, we compared APTT, PT, and plasma fibrinogen concentrations in the two patient groups in pre-intervention plasma samples. No statistical significant differences between the groups were found concerning APTT (37.26 ± 9.39 vs. 34.78 ± 6.95 s), PT (98.71 ± 16.53 vs. 98.75 ± 24.39 s), and plasma fibrinogen concentration (427.29 ± 99.75 vs. 400.40 ± 141.23 mg/dl). Identification of Patients at Risk for High- Degree Restenosis Using TEM-derived CTs To calculate the discrimination power between the two patient groups stepwise discriminant analysis was applied on our data. Only TEM-derived CT was significantly selected (p = 0.012), providing a sensitivity of 75.0 % and a specificity of 57.1 %. Twenty three out of 34 patients (67.6 %) were correctly classified by this CT value.
Furthermore, ROC curve analysis was applied on significantly different variables. Again the best discrimination result was achieved by CT with an area index of 0.709, a sensitivity of95 %, a specificity of 50 %, and 76.5 % correctly classified cases (26 of 34) at an optional cutoff value for the two patient groups of 444.5 s. Thus, under our experimental conditions, CT values shorter than 444.5 s identify patients at high risk (sensitivity = 95 %) for luminal narrowing in stents implanted in the SFA.
Discussion Previous studies suggest that thrombin, the pivotal enzyme in haemostasis, plays a critical role in the formation of ISR [4,5]. Thrombin is a powerful stimulator for the migration of macrophages and for the proliferation of smooth muscle cells thereby contributing to the later stages of the myoproliferative process in the pathogenesis of restenosis [19–21]. For example, high plasma levels of Heparin Cofactor II, a physiological inhibitor of thrombin actions, have been shown to be associated with reduced incidence of ISR after percutaneous coronary intervention [22]. Moreover, artificial thrombin inhibitors like recombinant hirudin or inogatran have been shown to reduce restenosis after angioplasty in animal models [23,24]. Therefore, in the present study, we investigated whether blood coagulation plays a role in the formation of ISR in PAD patients with stent angioplasty in the SFA. The coagulation status of 14 patients with low-degree restenosis was compared with that of 20 patients with high-degree restenosis three years after stent placement. The coagulation tests CAT, APTT, platelet aggregation, platelet adhesion, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two groups. Even levels of fibrinogen, a strong biochemical predictor of restenosis after coronary angioplasty [25–27], were comparable in both groups before, and, as indicated by same TEM-derived MCF values, three years after stent placement. However, the two TEM-derived values Coagulation Time (CT) and alpha angle were significantly deranged in the direction of hypercoagulability in the high-degree restenosis group. Whereas the alpha angle, representing the kinetics of fibrin build up and cross-linking, received only borderline significance (p = 0.045), CTs were markedly shorter in the high- degree restenosis group (p = 0.012).
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Sorensen et al. have shown that short CTs indicate fast thrombin generation [28]. Thus, a hypercoagulable state of patients with highdegree restenosis compared to patients with low-degree restenosis was detected in the present study by means of TEM employing activation with minute amounts of TF. To our knowledge, no studies exist to date reporting on the influence of blood coagulation/thrombin generation on the process of restenosis in PAD patients with stent implantation in the SFA. However, Schoebel et al. investigated the relevance of haemostasis on restenosis in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). They found that a hypercoagulable disease state prior to intervention characterizes a high-risk group prone for restenosis in clinically stable coronary artery disease [29]. In a similar study Salvioni et al. have shown a relation between thrombin activation soon after PTCA and late restenosis [30]. Gurbel et al. investigated the relation of ex vivo platelet reactivity, fibrin generation, and thrombin-induced clot strength to postdischarge ischemic events in patients undergoing percutaneous coronary intervention (PCI) over six month [1]. They found significantly higher platelet reactivity and fibrin formation, and, even more predictive, higher clot strength evaluated by means of TEM in patients with ischemic events after PCI. Thus, similar to our present study, a TEM derived parameter apparently allows identification of patients at high risk for thromboembolic events. In accordance, Wilson et al. have shown a significant correlation between hypercoagulability as determined by means of TEM and the development of deep venous thrombosis in 250 patients with femoral neck fracture [31]. In addition to these studies we show herein that patients with low-degree ISR are in a hypercoagulable state compared to patients with low-degree ISR not only before and immediately after, as shown by Schoebel et al. and Salvioni et al., but also three years after stent placement. In our previous study we have shown that patients with high risk for developing ISR and occlusion have elevated Apo B levels and low HDL cholesterol [32]. We assumed that platelet CD 36 interacts with oxidized lipoproteins, resulting in enhanced platelet reactivity [33]. However, the results in the present study do not support this assumption. Under our experimental conditions, platelet function was essentially the same in the low- and the high-degree ISR groups. The results from our present study suggest that the TEM-derived CT might be a suitable diagnostic parameter to reveal patients with a hypercoagulable state associated with elevated risk for formation of luminal narrowing and of ISR. However, CTs in our study were determined three years after stent placement. Thus, a further (prospective) study is needed to clarify whether TEM-derived CTs could actually serve as a predictor for ISR. This is in accordance with the call of Dai et al. for more prospective studies to clarify the predictive accuracy of TEM for postoperative thromboembolic events[34]. In this future study, TEM-derived CTs should be determined immediately before and at several time points after stent placement. It should be investigated whether short pre-angioplasty CTs correlate with enhanced formation of ISR. A limitation of our study is the small sample size as we evaluated only one vessel segment to minimize possible bias [35,36]. On the other hand, our patients are homogenous for the vessel segment treated and for the kind of endovascular treatment, which could be considered as the strength of our study. Further limitations of our study are that coagulability was not assessed during the time of neointimal formation and that platelet reactivity was determined when patients were off of clopidogrel. Neointimal formation is the major cause of ISR and high on-clopidogrel treatment is a major risk factor for recurrent ischemic events including target revascularization for ISR. Moreover, we did not perform arachidonic acid-induced platelet aggregation measurements in order to detect a possible influence of patients’ aspirin intake on platelet aggregation parameters. In conclusion, our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development
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