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Residual perfusion defects in patients with pulmonary embolism are related to impaired fibrinolytic capacity
Thrombosis Research 134 (2014) 737–741
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Regular Article
Residual perfusion defects in patients with pulmonary embolism are related to impaired fibrinolytic capacity Donatella Lami a,⁎, Anna Paola Cellai b, Emilia Antonucci a, Claudia Fiorillo c, Matteo Becatti c, Elisa Grifoni a, Caterina Cenci a, Rossella Marcucci a, Lucia Mannini b, Massimo Miniati a, Rosanna Abbate a, Domenico Prisco a a b c
Department of Experimental and Clinical Medicine, University of Florence, Florence Italy Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Department of Biochemical Sciences, University of Florence, Florence, Italy
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 4 July 2014 Accepted 10 July 2014 Available online 17 July 2014 Keywords: Pulmonary embolism Residual perfusion defects Clot lysis time Fibrinolysis
a b s t r a c t Background: Few studies investigated the relationship between fibrinolysis abnormalities and residual pulmonary perfusion defects after acute pulmonary embolism (PE). Objective: To assess the fibrinolytic profile in patients with prior PE in relation to the extent of scintigraphically detectable residual perfusion abnormalities. Patients and methods: We studied 71 consecutive patients with a prior episode of PE, who were examined after one year of the incident embolic event, and at least one month after anticoagulation withdrawal. They underwent lung scintigraphy to assess the recovery of pulmonary perfusion, echocardiography and chest radiography to look for signs of pulmonary hypertension. Clot formation and lysis were evaluated by two turbidimetric methods: Clot and Lysis Assay and Clot Lysis Time. We also measured the in vitro plasmin-mediated lysis of fibrin from purified fibrinogen, and the circulating levels of fibrinolytic inhibitors. The sample was split in two categories based on the extent of residual perfusion defects: b10% (n = 53), ≥10% (n = 18). Results: Patients with perfusion defects N 10% had significantly longer lysis time (p b 0.05), and higher levels of plasminogen activator inhibitor-1 (p b 0.01) than those with perfusion defects b 10%. The time interval between symptoms onset and PE diagnosis (time-to-diagnosis) was significantly longer in patients with perfusion defects N 10% than in the others (p = 0.005). In multivariate logistic regression, both lysis time and time-to-diagnosis were independently associated with perfusion defects N10% (p b 0.001). None of the sampled patients had echocardiographic or radiologic signs of pulmonary hypertension. Conclusion: Prolonged time-to-diagnosis and fibrinolysis imbalance are independent predictors of incomplete perfusion recovery after acute PE. © 2014 Elsevier Ltd. All rights reserved.
Introduction
In the present study, we investigated the relationship between fibrinolytic profile and extent of residual perfusion defects on lung scintigraphy in patients with a prior episode of acute PE. We measured the following parameters: 1) clot formation and lysis by two global functional tests; 2) fibrinolysis activators and inhibitors levels in plasma; 3) in vitro degradation of fibrin clots from purified fibrinogen.
Reportedly, some 15 to 30% of the patients with a prior episode of acute pulmonary embolism (PE) feature residual perfusion defects after a course of anticoagulant treatment [1–4]. Factors responsible for the persistence of such perfusion abnormalities are still unclear. Hypofibrinolysis is regarded as a risk factor for arterial and venous thrombosis, and the relative balance between clot formation and lysis is considered to reflect the thrombotic potential [5,6]. Recently, Undas et al. reported that clot properties (permeability and lysis) are altered in patients with incomplete recanalization of prior lower-limb deep vein thrombosis (DVT) [7]. Similarly, Cellai et al. described an altered fibrinolysis profile in patients with prior acute PE [8].
Methods
⁎ Corresponding author at: Thrombosis Centre, Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Viale Morgagni 85, 50134 Florence, Italy. Tel.: +39 055 7949421; fax: +39 055 7949418. E-mail address: [email protected] (D. Lami).
Sample The study sample included 71 consecutive patients with an objectively confirmed first episode of acute PE. They are part of a much larger
http://dx.doi.org/10.1016/j.thromres.2014.07.013 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Ethical Approval The study was approved by the local Ethics Committee (Azienda Ospedaliero-Universitaria Careggi, Florence, Italy). All the participants gave their written consent before entering the study.
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sample of patients who are currently referred to our institution for assessing (a) the recovery of pulmonary perfusion after acute PE, (b) the size and kinetics of the right heart cavities by echocardiography, and (c) the thrombophilic profile by standard laboratory tests. The baseline characteristics of 39 of the 71 patients with PE have been reported elsewhere [9]. Patients were interviewed by one of the coauthors (MM) using a standardized questionnaire [10]. This was done during the first visit at our outpatient clinic, which took place between four to six weeks after hospital discharge. “Time-to-diagnosis” was estimated as the interval between the onset of symptoms (as reported by the patient) and the objective testing for PE diagnosis (CT angiography). It is unlikely that a recall bias may have occurred because all the patients were interviewed shortly after their hospital discharge. All the patients had been treated with intravenous unfractioned heparin, or weight-adjusted low-molecular weight heparins followed by oral anticoagulant therapy with vitamin K antagonists with a target International Normalized Ratio (INR) of 2.5 (range 2.0–3.0). The following tests were obtained shortly before withdrawal of oral anticoagulation.
Blood Collection and Processing
Lung Scintigraphy
1) The time course of clot formation and lysis was assessed according to Carter et al. [18]., with some modifications.
Planar scintigraphic images (anterior, posterior, both lateral, and both posterior oblique views) were obtained after intravenous administration of human albumin macroaggregates labeled with 99mTechnetium (1.8x108 Bq) [11]. Images were examined by a nuclear medicine specialist, who was blinded to the time at which the lung scans were taken. Perfusion defects were estimated according to the method introduced by Meyer et al. [12], and validated against conventional pulmonary angiography. In the original paper, inter-rater agreement in assessing perfusion defects was N95% [12]. Such method was used by others to assess the restoration of pulmonary perfusion at varying times after the incident event [13–15]. Briefly, each lung lobe is attributed a weight according to regional blood flow as follows: right upper lobe, 0.18; right middle lobe, 0.12; right lower lobe, 0.25; left upper lobe, 0.13; lingula, 0.12; left lower lobe, 0.20. The perfusion of each lobe is estimated visually by means of a five-point score (0, 0.25, 0.5, 0.75, 1) where 0 means “not perfused” and 1 “normally perfused”. Visual estimates of perfusion are based on the combined evaluation of six scintigraphic views. Each lobar perfusion score is obtained by multiplying the weight assigned to the lobe by the estimated perfusion of that lobe. The overall score is the sum of the perfusion scores of the six lobes, and the percentage of pulmonary vascular obstruction is calculated as: (1–overall perfusion score)x100. According to the method we used, a perfusion scan is rated normal or near-normal if the pulmonary vascular obstruction score is ≤5% [12]. Based on a previous study [15], we set a cutpoint of ≥10% to indicate the persistence of clearly identifiable perfusion abnormalities. Scores of less than 10% reflect minor perfusion inhomogeneities rather than true defects [15].
Blood sampling was obtained not earlier than 12 months of the incident embolic event, and at least one month after discontinuation of anticoagulant therapy. After overnight fasting, blood samples were collected in Vacutainer tubes containing 0.109 M trisodium citrate (1:10, v/v, anticoagulant: blood) (Becton Dickinson, Plymouth, UK). Blood samples for plasminmediated lysis of fibrin, Clotting and Lysis Assay, thrombin activatable fibrinolysis inhibitor antigen (TAFI ag), plasminogen activator inhibitor-1 antigen (PAI-1 ag), tissue plasminogen activator antigen (t-PA ag), plasminogen (Plg), α-2-antiplasmin (α-2AP) were immediately centrifuged at 2000xg for 15 minutes at 4 °C; blood samples for fibrinogen and factor XIII assays (F XIII) were centrifuged at 2000xg for 15 minutes at room temperature. Plasma was separated and snap frozen in small aliquots that were stored at -80 °C until further assay. Clot Formation and Lysis We evaluated the clot structure and function using two sensitive turbimetric assays:
Postero-anterior and lateral digital chest radiographs were obtained at the time of lung scintigraphy. Chest films were examined by one of the coauthors (MM) for the presence of heart, pulmonary, or pleural abnormalities. Criteria used for identifying radiographic signs of pulmonary hypertension are given elsewhere [16].
a) Turbidimetric Clotting Assay: 25 μL of citrated plasma (in duplicate) was added to 75 μL assay buffer (0.05 mol/L Tris-HCl, 0.1 mol/L NaCl, pH 7.4), 50 μL of activation mix (final concentrations: 0.12 NIHU/mL thrombin [Sigma-Aldrich, St. Louis, MO, USA] and 7.5 mmol/L calcium in assay buffer) was added to each column of the 96-well plate using a multichannel pipette at 10 sec intervals (the time of addition of activation mix was recorded to enable plate reader times to be adjusted to the start of clot initiation). Plates were shaken and read at 405 nm every 12 sec for 1 hour in a TECAN infinite 200 microplate reader (Grodig, Austria). The following variables were determined from the turbidimetric clotting assay: lag time (Lag C), which represents the time at which sufficient protofibrils have formed to enable lateral aggregation, was taken as the time point at which an exponential increase in absorbance occurred; maximum absorbance (MaxAbs C) corrected for the Lag C absorbance; crude rate of clot formation (CRC) was derived from time and absorbance values. b) Turbidimetric Lysis Assay: 25 μL of citrated plasma (in duplicate) was added to 75 μL L assay buffer containing 60 ng/mL of t-PA (Actilyse; Boehringer Ingelheim, Germany) before addition of 50 μL of activation mix (final concentrations: 0.12 NIHU/mL thrombin [Sigma], and 7.5 mmol/L calcium in assay buffer); for this assay the plates were read at 405 nm every 12 sec for 1 hour and subsequently every 2 minutes for up to 4 hours. Time to 50% lysis (Lys50t0) was calculated as the time from initiation of clot formation to the time at which a 50% fall in absorbance from MaxAbs occurred. To facilitate the analysis of data from 96well plates, we used a simple algorithm built in R language to analyze a large amount of raw data. The inter-assay coefficients of variation (CV) were as follows: Lag C = 6%, CRC = 10%, MaxAbs C = 5 %, Lys50t0 = 11% (n = 14, for all variables). 2) Clot Lysis Time (CLT) was measured according to Lisman et al. [5] with some modifications as regards t-PA final concentration (50 ng/ml) and CLT calculation [19]. The intra-assay and interassay CVs were 4 % and 7% respectively.
Transthoracic Echocardiography
Endogenous Fibrinolysis Activators and Inhibitors
Transthoracic echocardiography (TTE) was obtained within two weeks of perfusion lung scintigraphy. Pulmonary hypertension was regarded as possible if the estimated pulmonary artery systolic pressure (PASP) exceeded 35 mmHg at rest [17].
Plasma TAFI ag levels were measured with a commercially available ELISA kit (Asserachrom TAFI, Diagnostica Stago, Asnieres, France). Plasma PAI-1 ag and t-PA ag levels were determined using commercially available ELISA kits (Asserachrom PAI-1 and Asserachrom t-PA,
Chest Radiography
D. Lami et al. / Thrombosis Research 134 (2014) 737–741
Diagnostica Stago, Asnieres, France). The measurements of factor XIII, α-2-antiplasmin, and plasminogen activity were performed with photometric determinations, using Behrichrom assays (Siemens, Marburg, Germany). Fibrinogen was evaluated by clotting assay according to Clauss. The intra-assay and inter-assay CVs for the above measurements ranged from 5% to 9%. Fibrinogen Purification, Fibrin Formation and Digestion with Plasmin The procedures of fibrinogen purification, fibrin formation, and degradation are described in detail elsewhere [9,20]. For in vitro degradation of fibrin clots, inter-assay CV was 10%. Statistical Analysis Continuous variables are presented as medians and interquartile range (IQR). Dichotomous variables were compared by Fisher’s exact test, and continuous variables by nonparametric Mann–Whitney test. Linear regression analysis was applied to assess the association of lysis time (Lys50t0) with the other laboratory data. With the exception of PAI-1 ag and t-PA ag, that required log transformation, all the continuous variables were normally distributed. Spearman’s correlation coefficients were used for evaluating the correlation between variables. We used stepwise linear regression to test the independent association between Lys50t0 and fibrinolytic parameters. For Lag C, Lys50t0, PAI-1 ag, and time-to-diagnosis, receiver operating characteristic curves were constructed to establish the cut-off values associated with the highest discriminating power between patients with residual perfusion defects ≥10% and all the others. Univariate logistic analysis was performed to assess the association of all the variables with the extent of residual perfusion defects on lung scintigraphy. The laboratory and clinical parameters that showed a statistically significant association with residual perfusion defects in univariate model (p b 0.05), and did not showed substantial correlations (r N 0.5) with other independent variables, were entered in multiple logistic regression analysis. Odds ratios (OR) are reported with their 95 % confidence interval (CI). Two-sided p-values b0.05 were considered statistically significant throughout. Statistical analysis was performed with SPSS (Statistical Package for Social Sciences, Chicago USA, software for Windows; version 14.0).
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Table 1 Baseline characteristics of the study sample. Residual perfusion defects on lung scan Variable
b10 % (n = 53)
≥10 % (n = 18)
P-value
Age, years Male BMI (Kg/m2) Current smokers Hypertension Dyslipidemia Diabetes Oral anticoagulation (months) Time-to-diagnosis (days) Transient risk factors⁎ Oral contraceptives Unprovoked PE Thrombophilia† Family history of VTE Type of VTE :DVT + PE Isolated PE
58 (43-70) 25 (47) 26 (23-30) 10 (19) 20 (38) 8 (15) 2 (4) 12 (8-16) 1 (1-150) 14 (26) 10 (19) 29 (55) 14 (26) 15 (28) 35 (66) 18 (34)
62 (49-71) 9 (50) 28 (23-31) 2 (11) 9 (50) 2 (11) 2 (11) 15 (10-25) 15 (1-90) 4 (22) 5 (28) 9 (50) 3 (17) 3 (17) 11 (61) 7 (39)
0.348 0.525 0.318 0.718 0.280 0.900 0.271 0.065 0.005 0.495 0.507 0.789 0.531 0.363 0.778
Data are numbers (%) or medians (interquartile range). BMI = body mass index. DVT = deep vein thrombosis. PE = pulmonary embolism. VTE = venous thromboembolism. ⁎ Recent major surgery or trauma, prolonged immobilization. † Factor V Leiden, prothrombin 20210A mutation, deficiency of protein C, protein S and antithrombin.
Lys50t0 was negatively correlated with Lag C (r = -0.480, p b 0.001), and positively correlated with PAI-1 ag (r = 0.661, p b 0.001). In univariate linear regression analysis, Lys50t0 was significantly correlated with log t-PA ag, TAFI ag, log PAI-1 ag, and Plg activity (Table 3). In stepwise multivariate analysis, PAI-1 and TAFI ag were retained in the final model at a statistically significant level (Table 3). PAI-1 and TAFI allowed explaining 44% of the clot lysis time variability (R2 = 0.44; p b 0.001). In univariate logistic regression analysis, perfusion defects ≥ 10% were significantly associated with Lag C (OR 4.3, p = 0.013), Lys50t0 (OR 4.6, p = 0.010), PAI-1 ag (OR 5.8, p = 0.003),) and time-todiagnosis (OR 7.2, p = 0.005). In multivariate logistic resgression, Lys50t0 and time-to-diagnosis were independently associated with residual perfusion defects ≥10% (Table 4).
Results Of the 71 patients with prior PE, 39 (55%) had a complete recovery of pulmonary perfusion at lung scintigraphy (obstruction score = 0), 14 (20%) had minor perfusion abnormalities (score 3 to 9%), and 18 (25%) featured residual perfusion defects affecting N 10% of the pulmonary vascular bed (range 10 to 37%). None of the 71 patients had PASP values equal to or greater than 35 mmHg on TTE. Similarly, none of them had chest radiographic abnormalities suggestive of chronic thromboembolic pulmonary hypertension (CTEPH). Therefore, performing right heart catheterization was deemed unethical. Table 1 shows the baseline characteristics of the study sample split as a function of the extent of residual perfusion defects (b 10% versus ≥10%). There was no significant difference across the two groups as regards age, sex, prevalence of unprovoked PE, thrombophilia, and cardiovascular or metabolic comorbidities. Time-to-diagnosis was significantly longer (p = 0.005) in patients with perfusion defects ≥10% than in the others. As shown in Table 2 and Fig. 1, patients with perfusion defects ≥10% featured the following statistically significant differences with respect to those with normal or near-normal scans: shorter Lag C, prolonged Lys50t0, and higher circulating levels of PAI-1 ag. No statistically significant differences were found between the two groups as regards CLT, degradation of fibrin β-chain, and other fibrinolytic parameters (Table 2).
Table 2 Fibrinolysis parameters in relation to residual perfusion defects. Residual perfusion defects on lung scan Variable Lag C (sec) CRC x 10-4 (au/sec) Max Abs C (au) Lys50t0 (sec) CLT (min) TAFI ag, microg/mL PAI-1 ag, ng/mL t-PA ag, ng/mL Plg act, % α-2-AP act, % FXIII act, % Fbg act, mg/dL Fbβ 1 h, % Fbβ 3 h, % Fbβ 6 h, %
b10 % (n = 53) 304 (275-324) 3.36 (2.87-4.19) 0.224 (0.181-0.259) 3664 (2864-4309) 63.8 (43-89) 9.0 (8.0-10.8) 14 (10-25) 7.8 (5.4-11.6) 103 (94-119) 106 (99-113) 122 (110-141) 313 (268-349) 84.6 (77.5-91.5) 58.7 (48.0-69.5) 33.0 (25.6-44.6)
≥10 % (n = 18) 266 (230-318) 3.90 (2.87-4.66) 0.239 (0.168-0.283) 4118 (3659-4576) 63.9 (52-82) 9.3 (8.3-10.4) 30 (17-51) 8.9 (5.9-12.4) 108 (103-125) 106 (101-113) 123 (112-134) 324 (300-401) 82.0 (77.0-88.9) 55.8 (49.0-64.3) 29.8 (21.0-43.0)
P-value 0.036 0.169 0.663 0.012 0.648 0.468 0.006 0.379 0.129 0.793 0.984 0.070 0.067 0.149 0.067
Data are medians (interquartile range). Ag = antigen. Act = activity. Au = absorbance units. Lag C = Lag Time. CRC = Crude Rate of Clot Formation. Max Abs C = Maximum Absorbance. Lys50t0 = Lysis time. CLT = Clot Lysis Time. TAFI = Thrombin Activatable Fibrinolysis Inhibitor. PAI-1 = Plasminogen Activator Inhibitor. t-PA = Tissue Plasminogen Activator. Plg = Plasminogen. α-2-AP = α-2-antiplasmin. Fbg = Fibrinogen. Fbβ 1 h, 3 h, 6 h,=amount of intact fibrin β-chains at 1, 3, 6 hours of digestion with plasmin.
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Fig. 1. Clot (a) and lysis (b) assay in 71 patients with prior pulmonary embolism split in two categories based on the extent of residual perfusion defects on lung scintigraphy: b10% black diamonds; ≥10% grey squares. Data are medians. OD = optical density; sec = seconds.
Discussion The results of our study can be summarized thus: (a) some 25% of the patients with a prior episode of acute PE feature persistent perfusion abnormalities on lung scintigraphy after one year of the incident event; (b) patients with incomplete perfusion recovery show a significantly longer lysis time, and significantly higher circulating levels of PAI-1 than those with normal or near-normal scans; (c) the time interval between onset of symptoms and definitive diagnosis of PE is significantly longer in patients with incomplete reperfusion than in the others. In addition, patients with residual perfusion defects ≥10% had a significantly faster protofibrils formation, a finding that has been reported by other investigators in patients with congestive heart failure and coronary artery disease [21,22]. By contrast, no difference was observed between the two groups as regards the rate of formation and the physical density of clots. In the clinical setting of DVT, Undas et al. [7] reported that lysis time is significantly longer in patients with residual venous occlusion with respect to those without, suggesting that hypofibrinolysis could play a role in determining incomplete venous recanalization. The present data lend support to the concept that impaired fibrinolysis may also have a negative impact on the restoration of pulmonary perfusion after acute PE. As opposed to lysis time, we found no significant difference in CLT between patients with incomplete perfusion recovery and those with normal or near-normal lung scans (Table 2). This is probably due to the different mechanisms of clot formation in the two assays. In CLT
Table 3 Association of fibrinolytic and anti-fibrinolytic parameters with lysis time (Lys50t0). Univariate Variable
b ± SE
Log t-PA ag TAFI ag log PAI-1 ag Plg act α-2-AP act FXIII act Fbg act Fbβ 1 h Fbβ 3 h Fbβ 6 h
1718 209 1755 22.7 23.1 9.4 2.6 -32.1 2.2 -18.9
± ± ± ± ± ± ± ± ± ±
Multivariate P-value
428 60 296 6.27 12.4 4.8 2.1 27.3 22.9 23.6
b0.001 0.001 b0.001 0.001 0.070 0.060 0.211 0.244 0.925 0.430
b = regression coefficient; SE = standard error. For other abbreviations see Table 2.
b ± SE
P-value
203 ± 52 1706 ± 278
b0.001 b0.001
assay, clotting is induced by tissue factor, whereas in lysis time assay it is activated by thrombin. Reportedly, fibrin clots from purified fibrinogen are resistant to plasmin-mediated lysis in both patients with CTEPH [9,20] and those with pulmonary hypertension other than thromboembolic [9]. Minor resistance of fibrin to lysis occurs in patients with prior PE and normal pulmonary artery pressure [8,9]. In the present study, we found no significant difference in the rate of fibrin breakdown in relation to the extent of residual perfusion abnormalites on lung scanning (Table 2). It should be considered, however, that fibrin clots are obtained after extensive purification of fibrinogen from other plasma components. Thus, the potential contribution of endogenous fibrinolytic proteins is abolished. In this regard, we found that (a) PAI-1 levels in plasma are significantly higher in patients with persistent perfusion abnormalities than in the others (Table 2), and (b) circulating levels of PAI-1 and TAFI account for nearly 45% of the variability in lysis time. The importance of elevated PAI-1 and TAFI as risk factors for venous thromboembolism has been emphasized in previous studies [8,23]. Also, Lang and coworkers showed that PAI-1 is over-exspressed in pulmonary clots from patients with CTEPH, thereby suggesting a potential role in the stabilization of vascular thrombi [24]. Thromboembolic complications are also associated with the formation of compact fibrin clots which are resistant to lysis [25]. In fact, the architecture of the fibrin clot (fiber thickness and pore size) is thought to affect the efficiency of fibrinolysis [26]. In multivariate logistic regression, we found that lysis time and timeto-diagnosis are both independent predictors of incomplete perfusion recovery after PE (Table 4). The latter finding is in keeping with the observation of Sanchez et al. that a prolonged time interval between onset of symptoms and diagnosis of PE is an indipendent risk factor for persistent perfusion defects [27]. Over a median follow-up time of one year, 73 (29%) of 254 patients with prior PE had persistent ventilationperfusion mismatch in at least two lung segments [27]. These patients featured significantly higher PASP values on TTE, and were more likely to have dyspnea on exertion than the others [27].
Table 4 Multivariate logistic regression analysis. Variable
Odds Ratio
95% CI
P-value
Lys50t0 (sec)* Time-to-diagnosis (days)†
7.11 11.07
1.78-28.35 2.41-50.74
0.005 0.002
Lys50t0 = lysis time at 50%. CI = confidence interval. *Cutpoint: 3878 sec. †Cutpoint: 2 days.
D. Lami et al. / Thrombosis Research 134 (2014) 737–741
The data of Sanchez and ours underscore the importance of a prompt diagnosis of PE. This in turn depends on the physician’s ability to raise the suspicion of the disease on the basis of relevent presenting symptoms and signs [10]. Study Limitations First, the study sample is relatively small, and originates from a single referral center. Broader studies are, therefore, needed to establish the impact of an altered fibrinolytic profile on recovery of pulmonary perfusion after acute PE. Second, the fibrinolytic profile was assessed after the thromboembolic event had occurred, so that we cannot firmly establish a causal relationship between fibrinolysis impairment and persistent perfusion abnormalities in the lung. Third, the estimation of the time interval between symptoms onset and PE diagnosis cannot be absolutely precise for the perception of symptoms may vary substantially from one patient to another. Conclusion In conclusion, our data indicate that prolonged time between symptoms onset and PE diagnosis and fibrinolytic imbalance are both independent predictors of incomplete recovery of pulmonary perfusion after acute PE. Authors Contribution Conception and design: DL, APC, RA, DP. Collection and analysis of data: DL, APC, CF, MB, MM. Statistical analysis: DL. Drafting the manuscript: DL, APC, MM. Critical review of the manuscript: EA, RM, LM, RA, DP. References [1] Stein PD, Yaekoub AY, Matta F, Janjua M, Patel RM, Goodman LR, et al. Resolution of pulmonary embolism on CT pulmonary angiography. Am J Roentgenol 2010; 194:1263–8. [2] Cosmi B, Nijkeuter M, Valentino M, Huisman MV, Barozzi L, Palareti G. Residual emboli on lung perfusion scan or multidetector computed tomography after a first episode of acute pulmonary embolism. Intern Emerg Med 2011;6:521–8. [3] Alonso-Martínez JL, Anniccherico-Sánchez FJ, Urbieta-Echezarreta MA, GarcíaSanchotena JL, Herrero HG. Residual pulmonary thromboemboli after acute pulmonary embolism. Eur J Intern Med 2012;23:379–83. [4] Golpe R, de Llano LA, Castro-Añón O, Vázquez-Caruncho M, González-Juanatey C, Fariñas MC. Long-term outcome of patients with persistent vascular obstruction on computed tomography pulmonary angiography 6 months after acute pulmonary embolism. Acta Radiol 2012;53:728–31. [5] Lisman T, de Groot PG, Meijers JCM, Rosendaal FR. Reduced plasma fibrinolytic potential is a risk factor for venous thrombosis. Blood 2005;105:1102–5. [6] Guimarães AH, de Bruijne EL, Lisman T, Dippel DW, Deckers JW, Poldermans D, et al. Hypofibrinolysis is a risk factor for arterial thrombosis at young age. Br J Haematol 2009;145:115–20.
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[7] Undas A, Cieśla-Dul M, Drążkiewicz T, Sadowski J. Altered fibrin clot properties are associated with residual vein obstruction: effects of lipoprotein(a) and apolipoprotein(a) isoform. Thromb Res 2012;130:184–7. [8] Cellai AP, Lami D, Antonucci E, Fiorillo C, Becatti M, Olimpieri B, et al. Fibrinolytic inhibitors and fibrin characteristics determine a hypofibrinolytic state in patients with pulmonary embolism. Thromb Haemost 2013;109:565–7. [9] Miniati M, Fiorillo C, Becatti M, Monti S, Bottai M, Marini C, et al. Fibrin resistance to lysis in patients with pulmonary hypertension other than thromboembolic. Am J Respir Crit Care Med 2010;181:992–6. [10] Miniati M, Cenci C, Monti S, Poli D. Clinical presentation of acute pulmonary embolism. A survey of 800 cases. PLoS ONE 2012;7:e30891. [11] Miniati M, Pistolesi M, Marini C, Di Ricco G, Formichi B, Prediletto R, et al. Value of perfusion lung scan in the diagnosis of pulmonary embolism: results of the Prospective Investigative Study of Acute Pulmonary Embolism Diagnosis (PISA-PED). Am J Respir Crit Care Med 1996;154:1387–93. [12] Meyer G, Collignon MA, Guinet F, Jeffrey AA, Barritault L, Sors H. Comparison of perfusion lung scanning and angiography in the estimation of vascular obstruction in acute pulmonary embolism. Eur J Nucl Med 1990;17:315–9. [13] Simonneau G, Sors H, Charbonnier B, Page Y, Laaban JP, Azarian R, The THESEE Study Group, et al. A comparison of low molecular weight heparin with unfractionated heparin for acute pulmonary embolism. N Engl J Med 1997;337:663–9. [14] Wartski M, Collignon MA, for the THESEE Study Group. Incomplete recovery of lung perfusion after 3 months in patients with acute pulmonary embolism treated with antithrombotic agents. J Nucl Med 2000;41:1043–8. [15] Miniati M, Monti S, Bottai M, Scoscia E, Bauleo C, Tonelli L, et al. Survival and restoration of pulmonary perfusion in a long-term follow-up of patients after acute pulmonary embolism. Medicine (Baltimore) 2006;85:253–62. [16] Miniati M, Monti S, Airò E, Pancani R, Bauleo C, Formichi B, et al. Accuracy of chest radiography in predicting pulmonary hypertension. Thromb Res 2014;133:345–51. [17] Galiè N, Hoeper MM, Humbert M, Torbicki A, Vachiery JL, Barbera JA, et al. The Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS), endorsed by the International Society of Heart and Lung Transplantation (ISHLT) Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Heart J 2009; 30:2495–537. [18] Carter AM, Cymbalista CM, Spector TD, Grant PJ, Heritability of Clot Formation, Morphology, and Lysis: The EuroCLOT Study, on behalf of the Euro CLOT investigators. Arterioscler Thromb Vasc Biol 2007;27:2783–9. [19] Cellai AP, Lami D, Magi A, Liotta AA, Rogolino A, Antonucci E, et al. Assessment of fibrinolytic activity by measuring the lysis time of a tissue-factor-induced clot: a feasibility evaluation. Clin Appl Thromb Hemost 2010;16:337–44. [20] Morris TA, Marsh JJ, Chiles PG, Auger WR, Fedullo PF, Woods Jr VL. Fibrin derived from patients with chronic thromboembolic pulmonary hypertension is resistant to lysis. Am J Respir Crit Care Med 2006;173:1270–5. [21] Palka I, Nessler J, Nessler B, Piwowarska W, Tracz W, Undas A. Altered fibrin clot properties in patients with chronic heart failure and sinus rhythm: a novel prothrombotic mechanism. Heart 2010;96:1114–8. [22] Mills JD, Ariëns RA, Mansfield MW, Grant PJ. Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. Circulation 2002;106:1938–42. [23] Meltzer ME, Lisman T, de Groot PG, Meijers JC, le Cessie S, Doggen CJ, et al. Venous thrombosis risk associated with plasma hypofibrinolysis is explained by elevated plasma levels of TAFI and PAI-1. Blood 2010;116:113–21. [24] Lang IM, Marsh JJ, Olman MA, Moser KM, Loskutoff DJ, Schleef RR. Expression of type 1 plasminogen activator inhibitor in chronic pulmonary thromboemboli. Circulation 1994;89:2715–21. [25] Undas A, Zawilska K, Ciesla-Dul M, Lehmann-Kopydłowska A, Skubiszak A, Ciepłuch K, et al. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood 2009;114:4272–8. [26] Blombäck B. Fibrinogen and fibrin–proteins with complex roles in hemostasis and thrombosis. Thromb Res 1996;83:1–75. [27] Sanchez O, Helley D, Couchon S, Roux A, Delaval A, Trinquart L, et al. Perfusion defects after pulmonary embolism: risk factors and clinical significance. J Thromb Haemost 2010;8:1248–55.
Contents lists available at ScienceDirect
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Regular Article
Residual perfusion defects in patients with pulmonary embolism are related to impaired fibrinolytic capacity Donatella Lami a,⁎, Anna Paola Cellai b, Emilia Antonucci a, Claudia Fiorillo c, Matteo Becatti c, Elisa Grifoni a, Caterina Cenci a, Rossella Marcucci a, Lucia Mannini b, Massimo Miniati a, Rosanna Abbate a, Domenico Prisco a a b c
Department of Experimental and Clinical Medicine, University of Florence, Florence Italy Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Department of Biochemical Sciences, University of Florence, Florence, Italy
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 4 July 2014 Accepted 10 July 2014 Available online 17 July 2014 Keywords: Pulmonary embolism Residual perfusion defects Clot lysis time Fibrinolysis
a b s t r a c t Background: Few studies investigated the relationship between fibrinolysis abnormalities and residual pulmonary perfusion defects after acute pulmonary embolism (PE). Objective: To assess the fibrinolytic profile in patients with prior PE in relation to the extent of scintigraphically detectable residual perfusion abnormalities. Patients and methods: We studied 71 consecutive patients with a prior episode of PE, who were examined after one year of the incident embolic event, and at least one month after anticoagulation withdrawal. They underwent lung scintigraphy to assess the recovery of pulmonary perfusion, echocardiography and chest radiography to look for signs of pulmonary hypertension. Clot formation and lysis were evaluated by two turbidimetric methods: Clot and Lysis Assay and Clot Lysis Time. We also measured the in vitro plasmin-mediated lysis of fibrin from purified fibrinogen, and the circulating levels of fibrinolytic inhibitors. The sample was split in two categories based on the extent of residual perfusion defects: b10% (n = 53), ≥10% (n = 18). Results: Patients with perfusion defects N 10% had significantly longer lysis time (p b 0.05), and higher levels of plasminogen activator inhibitor-1 (p b 0.01) than those with perfusion defects b 10%. The time interval between symptoms onset and PE diagnosis (time-to-diagnosis) was significantly longer in patients with perfusion defects N 10% than in the others (p = 0.005). In multivariate logistic regression, both lysis time and time-to-diagnosis were independently associated with perfusion defects N10% (p b 0.001). None of the sampled patients had echocardiographic or radiologic signs of pulmonary hypertension. Conclusion: Prolonged time-to-diagnosis and fibrinolysis imbalance are independent predictors of incomplete perfusion recovery after acute PE. © 2014 Elsevier Ltd. All rights reserved.
Introduction
In the present study, we investigated the relationship between fibrinolytic profile and extent of residual perfusion defects on lung scintigraphy in patients with a prior episode of acute PE. We measured the following parameters: 1) clot formation and lysis by two global functional tests; 2) fibrinolysis activators and inhibitors levels in plasma; 3) in vitro degradation of fibrin clots from purified fibrinogen.
Reportedly, some 15 to 30% of the patients with a prior episode of acute pulmonary embolism (PE) feature residual perfusion defects after a course of anticoagulant treatment [1–4]. Factors responsible for the persistence of such perfusion abnormalities are still unclear. Hypofibrinolysis is regarded as a risk factor for arterial and venous thrombosis, and the relative balance between clot formation and lysis is considered to reflect the thrombotic potential [5,6]. Recently, Undas et al. reported that clot properties (permeability and lysis) are altered in patients with incomplete recanalization of prior lower-limb deep vein thrombosis (DVT) [7]. Similarly, Cellai et al. described an altered fibrinolysis profile in patients with prior acute PE [8].
Methods
⁎ Corresponding author at: Thrombosis Centre, Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Viale Morgagni 85, 50134 Florence, Italy. Tel.: +39 055 7949421; fax: +39 055 7949418. E-mail address: [email protected] (D. Lami).
Sample The study sample included 71 consecutive patients with an objectively confirmed first episode of acute PE. They are part of a much larger
http://dx.doi.org/10.1016/j.thromres.2014.07.013 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Ethical Approval The study was approved by the local Ethics Committee (Azienda Ospedaliero-Universitaria Careggi, Florence, Italy). All the participants gave their written consent before entering the study.
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sample of patients who are currently referred to our institution for assessing (a) the recovery of pulmonary perfusion after acute PE, (b) the size and kinetics of the right heart cavities by echocardiography, and (c) the thrombophilic profile by standard laboratory tests. The baseline characteristics of 39 of the 71 patients with PE have been reported elsewhere [9]. Patients were interviewed by one of the coauthors (MM) using a standardized questionnaire [10]. This was done during the first visit at our outpatient clinic, which took place between four to six weeks after hospital discharge. “Time-to-diagnosis” was estimated as the interval between the onset of symptoms (as reported by the patient) and the objective testing for PE diagnosis (CT angiography). It is unlikely that a recall bias may have occurred because all the patients were interviewed shortly after their hospital discharge. All the patients had been treated with intravenous unfractioned heparin, or weight-adjusted low-molecular weight heparins followed by oral anticoagulant therapy with vitamin K antagonists with a target International Normalized Ratio (INR) of 2.5 (range 2.0–3.0). The following tests were obtained shortly before withdrawal of oral anticoagulation.
Blood Collection and Processing
Lung Scintigraphy
1) The time course of clot formation and lysis was assessed according to Carter et al. [18]., with some modifications.
Planar scintigraphic images (anterior, posterior, both lateral, and both posterior oblique views) were obtained after intravenous administration of human albumin macroaggregates labeled with 99mTechnetium (1.8x108 Bq) [11]. Images were examined by a nuclear medicine specialist, who was blinded to the time at which the lung scans were taken. Perfusion defects were estimated according to the method introduced by Meyer et al. [12], and validated against conventional pulmonary angiography. In the original paper, inter-rater agreement in assessing perfusion defects was N95% [12]. Such method was used by others to assess the restoration of pulmonary perfusion at varying times after the incident event [13–15]. Briefly, each lung lobe is attributed a weight according to regional blood flow as follows: right upper lobe, 0.18; right middle lobe, 0.12; right lower lobe, 0.25; left upper lobe, 0.13; lingula, 0.12; left lower lobe, 0.20. The perfusion of each lobe is estimated visually by means of a five-point score (0, 0.25, 0.5, 0.75, 1) where 0 means “not perfused” and 1 “normally perfused”. Visual estimates of perfusion are based on the combined evaluation of six scintigraphic views. Each lobar perfusion score is obtained by multiplying the weight assigned to the lobe by the estimated perfusion of that lobe. The overall score is the sum of the perfusion scores of the six lobes, and the percentage of pulmonary vascular obstruction is calculated as: (1–overall perfusion score)x100. According to the method we used, a perfusion scan is rated normal or near-normal if the pulmonary vascular obstruction score is ≤5% [12]. Based on a previous study [15], we set a cutpoint of ≥10% to indicate the persistence of clearly identifiable perfusion abnormalities. Scores of less than 10% reflect minor perfusion inhomogeneities rather than true defects [15].
Blood sampling was obtained not earlier than 12 months of the incident embolic event, and at least one month after discontinuation of anticoagulant therapy. After overnight fasting, blood samples were collected in Vacutainer tubes containing 0.109 M trisodium citrate (1:10, v/v, anticoagulant: blood) (Becton Dickinson, Plymouth, UK). Blood samples for plasminmediated lysis of fibrin, Clotting and Lysis Assay, thrombin activatable fibrinolysis inhibitor antigen (TAFI ag), plasminogen activator inhibitor-1 antigen (PAI-1 ag), tissue plasminogen activator antigen (t-PA ag), plasminogen (Plg), α-2-antiplasmin (α-2AP) were immediately centrifuged at 2000xg for 15 minutes at 4 °C; blood samples for fibrinogen and factor XIII assays (F XIII) were centrifuged at 2000xg for 15 minutes at room temperature. Plasma was separated and snap frozen in small aliquots that were stored at -80 °C until further assay. Clot Formation and Lysis We evaluated the clot structure and function using two sensitive turbimetric assays:
Postero-anterior and lateral digital chest radiographs were obtained at the time of lung scintigraphy. Chest films were examined by one of the coauthors (MM) for the presence of heart, pulmonary, or pleural abnormalities. Criteria used for identifying radiographic signs of pulmonary hypertension are given elsewhere [16].
a) Turbidimetric Clotting Assay: 25 μL of citrated plasma (in duplicate) was added to 75 μL assay buffer (0.05 mol/L Tris-HCl, 0.1 mol/L NaCl, pH 7.4), 50 μL of activation mix (final concentrations: 0.12 NIHU/mL thrombin [Sigma-Aldrich, St. Louis, MO, USA] and 7.5 mmol/L calcium in assay buffer) was added to each column of the 96-well plate using a multichannel pipette at 10 sec intervals (the time of addition of activation mix was recorded to enable plate reader times to be adjusted to the start of clot initiation). Plates were shaken and read at 405 nm every 12 sec for 1 hour in a TECAN infinite 200 microplate reader (Grodig, Austria). The following variables were determined from the turbidimetric clotting assay: lag time (Lag C), which represents the time at which sufficient protofibrils have formed to enable lateral aggregation, was taken as the time point at which an exponential increase in absorbance occurred; maximum absorbance (MaxAbs C) corrected for the Lag C absorbance; crude rate of clot formation (CRC) was derived from time and absorbance values. b) Turbidimetric Lysis Assay: 25 μL of citrated plasma (in duplicate) was added to 75 μL L assay buffer containing 60 ng/mL of t-PA (Actilyse; Boehringer Ingelheim, Germany) before addition of 50 μL of activation mix (final concentrations: 0.12 NIHU/mL thrombin [Sigma], and 7.5 mmol/L calcium in assay buffer); for this assay the plates were read at 405 nm every 12 sec for 1 hour and subsequently every 2 minutes for up to 4 hours. Time to 50% lysis (Lys50t0) was calculated as the time from initiation of clot formation to the time at which a 50% fall in absorbance from MaxAbs occurred. To facilitate the analysis of data from 96well plates, we used a simple algorithm built in R language to analyze a large amount of raw data. The inter-assay coefficients of variation (CV) were as follows: Lag C = 6%, CRC = 10%, MaxAbs C = 5 %, Lys50t0 = 11% (n = 14, for all variables). 2) Clot Lysis Time (CLT) was measured according to Lisman et al. [5] with some modifications as regards t-PA final concentration (50 ng/ml) and CLT calculation [19]. The intra-assay and interassay CVs were 4 % and 7% respectively.
Transthoracic Echocardiography
Endogenous Fibrinolysis Activators and Inhibitors
Transthoracic echocardiography (TTE) was obtained within two weeks of perfusion lung scintigraphy. Pulmonary hypertension was regarded as possible if the estimated pulmonary artery systolic pressure (PASP) exceeded 35 mmHg at rest [17].
Plasma TAFI ag levels were measured with a commercially available ELISA kit (Asserachrom TAFI, Diagnostica Stago, Asnieres, France). Plasma PAI-1 ag and t-PA ag levels were determined using commercially available ELISA kits (Asserachrom PAI-1 and Asserachrom t-PA,
Chest Radiography
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Diagnostica Stago, Asnieres, France). The measurements of factor XIII, α-2-antiplasmin, and plasminogen activity were performed with photometric determinations, using Behrichrom assays (Siemens, Marburg, Germany). Fibrinogen was evaluated by clotting assay according to Clauss. The intra-assay and inter-assay CVs for the above measurements ranged from 5% to 9%. Fibrinogen Purification, Fibrin Formation and Digestion with Plasmin The procedures of fibrinogen purification, fibrin formation, and degradation are described in detail elsewhere [9,20]. For in vitro degradation of fibrin clots, inter-assay CV was 10%. Statistical Analysis Continuous variables are presented as medians and interquartile range (IQR). Dichotomous variables were compared by Fisher’s exact test, and continuous variables by nonparametric Mann–Whitney test. Linear regression analysis was applied to assess the association of lysis time (Lys50t0) with the other laboratory data. With the exception of PAI-1 ag and t-PA ag, that required log transformation, all the continuous variables were normally distributed. Spearman’s correlation coefficients were used for evaluating the correlation between variables. We used stepwise linear regression to test the independent association between Lys50t0 and fibrinolytic parameters. For Lag C, Lys50t0, PAI-1 ag, and time-to-diagnosis, receiver operating characteristic curves were constructed to establish the cut-off values associated with the highest discriminating power between patients with residual perfusion defects ≥10% and all the others. Univariate logistic analysis was performed to assess the association of all the variables with the extent of residual perfusion defects on lung scintigraphy. The laboratory and clinical parameters that showed a statistically significant association with residual perfusion defects in univariate model (p b 0.05), and did not showed substantial correlations (r N 0.5) with other independent variables, were entered in multiple logistic regression analysis. Odds ratios (OR) are reported with their 95 % confidence interval (CI). Two-sided p-values b0.05 were considered statistically significant throughout. Statistical analysis was performed with SPSS (Statistical Package for Social Sciences, Chicago USA, software for Windows; version 14.0).
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Table 1 Baseline characteristics of the study sample. Residual perfusion defects on lung scan Variable
b10 % (n = 53)
≥10 % (n = 18)
P-value
Age, years Male BMI (Kg/m2) Current smokers Hypertension Dyslipidemia Diabetes Oral anticoagulation (months) Time-to-diagnosis (days) Transient risk factors⁎ Oral contraceptives Unprovoked PE Thrombophilia† Family history of VTE Type of VTE :DVT + PE Isolated PE
58 (43-70) 25 (47) 26 (23-30) 10 (19) 20 (38) 8 (15) 2 (4) 12 (8-16) 1 (1-150) 14 (26) 10 (19) 29 (55) 14 (26) 15 (28) 35 (66) 18 (34)
62 (49-71) 9 (50) 28 (23-31) 2 (11) 9 (50) 2 (11) 2 (11) 15 (10-25) 15 (1-90) 4 (22) 5 (28) 9 (50) 3 (17) 3 (17) 11 (61) 7 (39)
0.348 0.525 0.318 0.718 0.280 0.900 0.271 0.065 0.005 0.495 0.507 0.789 0.531 0.363 0.778
Data are numbers (%) or medians (interquartile range). BMI = body mass index. DVT = deep vein thrombosis. PE = pulmonary embolism. VTE = venous thromboembolism. ⁎ Recent major surgery or trauma, prolonged immobilization. † Factor V Leiden, prothrombin 20210A mutation, deficiency of protein C, protein S and antithrombin.
Lys50t0 was negatively correlated with Lag C (r = -0.480, p b 0.001), and positively correlated with PAI-1 ag (r = 0.661, p b 0.001). In univariate linear regression analysis, Lys50t0 was significantly correlated with log t-PA ag, TAFI ag, log PAI-1 ag, and Plg activity (Table 3). In stepwise multivariate analysis, PAI-1 and TAFI ag were retained in the final model at a statistically significant level (Table 3). PAI-1 and TAFI allowed explaining 44% of the clot lysis time variability (R2 = 0.44; p b 0.001). In univariate logistic regression analysis, perfusion defects ≥ 10% were significantly associated with Lag C (OR 4.3, p = 0.013), Lys50t0 (OR 4.6, p = 0.010), PAI-1 ag (OR 5.8, p = 0.003),) and time-todiagnosis (OR 7.2, p = 0.005). In multivariate logistic resgression, Lys50t0 and time-to-diagnosis were independently associated with residual perfusion defects ≥10% (Table 4).
Results Of the 71 patients with prior PE, 39 (55%) had a complete recovery of pulmonary perfusion at lung scintigraphy (obstruction score = 0), 14 (20%) had minor perfusion abnormalities (score 3 to 9%), and 18 (25%) featured residual perfusion defects affecting N 10% of the pulmonary vascular bed (range 10 to 37%). None of the 71 patients had PASP values equal to or greater than 35 mmHg on TTE. Similarly, none of them had chest radiographic abnormalities suggestive of chronic thromboembolic pulmonary hypertension (CTEPH). Therefore, performing right heart catheterization was deemed unethical. Table 1 shows the baseline characteristics of the study sample split as a function of the extent of residual perfusion defects (b 10% versus ≥10%). There was no significant difference across the two groups as regards age, sex, prevalence of unprovoked PE, thrombophilia, and cardiovascular or metabolic comorbidities. Time-to-diagnosis was significantly longer (p = 0.005) in patients with perfusion defects ≥10% than in the others. As shown in Table 2 and Fig. 1, patients with perfusion defects ≥10% featured the following statistically significant differences with respect to those with normal or near-normal scans: shorter Lag C, prolonged Lys50t0, and higher circulating levels of PAI-1 ag. No statistically significant differences were found between the two groups as regards CLT, degradation of fibrin β-chain, and other fibrinolytic parameters (Table 2).
Table 2 Fibrinolysis parameters in relation to residual perfusion defects. Residual perfusion defects on lung scan Variable Lag C (sec) CRC x 10-4 (au/sec) Max Abs C (au) Lys50t0 (sec) CLT (min) TAFI ag, microg/mL PAI-1 ag, ng/mL t-PA ag, ng/mL Plg act, % α-2-AP act, % FXIII act, % Fbg act, mg/dL Fbβ 1 h, % Fbβ 3 h, % Fbβ 6 h, %
b10 % (n = 53) 304 (275-324) 3.36 (2.87-4.19) 0.224 (0.181-0.259) 3664 (2864-4309) 63.8 (43-89) 9.0 (8.0-10.8) 14 (10-25) 7.8 (5.4-11.6) 103 (94-119) 106 (99-113) 122 (110-141) 313 (268-349) 84.6 (77.5-91.5) 58.7 (48.0-69.5) 33.0 (25.6-44.6)
≥10 % (n = 18) 266 (230-318) 3.90 (2.87-4.66) 0.239 (0.168-0.283) 4118 (3659-4576) 63.9 (52-82) 9.3 (8.3-10.4) 30 (17-51) 8.9 (5.9-12.4) 108 (103-125) 106 (101-113) 123 (112-134) 324 (300-401) 82.0 (77.0-88.9) 55.8 (49.0-64.3) 29.8 (21.0-43.0)
P-value 0.036 0.169 0.663 0.012 0.648 0.468 0.006 0.379 0.129 0.793 0.984 0.070 0.067 0.149 0.067
Data are medians (interquartile range). Ag = antigen. Act = activity. Au = absorbance units. Lag C = Lag Time. CRC = Crude Rate of Clot Formation. Max Abs C = Maximum Absorbance. Lys50t0 = Lysis time. CLT = Clot Lysis Time. TAFI = Thrombin Activatable Fibrinolysis Inhibitor. PAI-1 = Plasminogen Activator Inhibitor. t-PA = Tissue Plasminogen Activator. Plg = Plasminogen. α-2-AP = α-2-antiplasmin. Fbg = Fibrinogen. Fbβ 1 h, 3 h, 6 h,=amount of intact fibrin β-chains at 1, 3, 6 hours of digestion with plasmin.
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Fig. 1. Clot (a) and lysis (b) assay in 71 patients with prior pulmonary embolism split in two categories based on the extent of residual perfusion defects on lung scintigraphy: b10% black diamonds; ≥10% grey squares. Data are medians. OD = optical density; sec = seconds.
Discussion The results of our study can be summarized thus: (a) some 25% of the patients with a prior episode of acute PE feature persistent perfusion abnormalities on lung scintigraphy after one year of the incident event; (b) patients with incomplete perfusion recovery show a significantly longer lysis time, and significantly higher circulating levels of PAI-1 than those with normal or near-normal scans; (c) the time interval between onset of symptoms and definitive diagnosis of PE is significantly longer in patients with incomplete reperfusion than in the others. In addition, patients with residual perfusion defects ≥10% had a significantly faster protofibrils formation, a finding that has been reported by other investigators in patients with congestive heart failure and coronary artery disease [21,22]. By contrast, no difference was observed between the two groups as regards the rate of formation and the physical density of clots. In the clinical setting of DVT, Undas et al. [7] reported that lysis time is significantly longer in patients with residual venous occlusion with respect to those without, suggesting that hypofibrinolysis could play a role in determining incomplete venous recanalization. The present data lend support to the concept that impaired fibrinolysis may also have a negative impact on the restoration of pulmonary perfusion after acute PE. As opposed to lysis time, we found no significant difference in CLT between patients with incomplete perfusion recovery and those with normal or near-normal lung scans (Table 2). This is probably due to the different mechanisms of clot formation in the two assays. In CLT
Table 3 Association of fibrinolytic and anti-fibrinolytic parameters with lysis time (Lys50t0). Univariate Variable
b ± SE
Log t-PA ag TAFI ag log PAI-1 ag Plg act α-2-AP act FXIII act Fbg act Fbβ 1 h Fbβ 3 h Fbβ 6 h
1718 209 1755 22.7 23.1 9.4 2.6 -32.1 2.2 -18.9
± ± ± ± ± ± ± ± ± ±
Multivariate P-value
428 60 296 6.27 12.4 4.8 2.1 27.3 22.9 23.6
b0.001 0.001 b0.001 0.001 0.070 0.060 0.211 0.244 0.925 0.430
b = regression coefficient; SE = standard error. For other abbreviations see Table 2.
b ± SE
P-value
203 ± 52 1706 ± 278
b0.001 b0.001
assay, clotting is induced by tissue factor, whereas in lysis time assay it is activated by thrombin. Reportedly, fibrin clots from purified fibrinogen are resistant to plasmin-mediated lysis in both patients with CTEPH [9,20] and those with pulmonary hypertension other than thromboembolic [9]. Minor resistance of fibrin to lysis occurs in patients with prior PE and normal pulmonary artery pressure [8,9]. In the present study, we found no significant difference in the rate of fibrin breakdown in relation to the extent of residual perfusion abnormalites on lung scanning (Table 2). It should be considered, however, that fibrin clots are obtained after extensive purification of fibrinogen from other plasma components. Thus, the potential contribution of endogenous fibrinolytic proteins is abolished. In this regard, we found that (a) PAI-1 levels in plasma are significantly higher in patients with persistent perfusion abnormalities than in the others (Table 2), and (b) circulating levels of PAI-1 and TAFI account for nearly 45% of the variability in lysis time. The importance of elevated PAI-1 and TAFI as risk factors for venous thromboembolism has been emphasized in previous studies [8,23]. Also, Lang and coworkers showed that PAI-1 is over-exspressed in pulmonary clots from patients with CTEPH, thereby suggesting a potential role in the stabilization of vascular thrombi [24]. Thromboembolic complications are also associated with the formation of compact fibrin clots which are resistant to lysis [25]. In fact, the architecture of the fibrin clot (fiber thickness and pore size) is thought to affect the efficiency of fibrinolysis [26]. In multivariate logistic regression, we found that lysis time and timeto-diagnosis are both independent predictors of incomplete perfusion recovery after PE (Table 4). The latter finding is in keeping with the observation of Sanchez et al. that a prolonged time interval between onset of symptoms and diagnosis of PE is an indipendent risk factor for persistent perfusion defects [27]. Over a median follow-up time of one year, 73 (29%) of 254 patients with prior PE had persistent ventilationperfusion mismatch in at least two lung segments [27]. These patients featured significantly higher PASP values on TTE, and were more likely to have dyspnea on exertion than the others [27].
Table 4 Multivariate logistic regression analysis. Variable
Odds Ratio
95% CI
P-value
Lys50t0 (sec)* Time-to-diagnosis (days)†
7.11 11.07
1.78-28.35 2.41-50.74
0.005 0.002
Lys50t0 = lysis time at 50%. CI = confidence interval. *Cutpoint: 3878 sec. †Cutpoint: 2 days.
D. Lami et al. / Thrombosis Research 134 (2014) 737–741
The data of Sanchez and ours underscore the importance of a prompt diagnosis of PE. This in turn depends on the physician’s ability to raise the suspicion of the disease on the basis of relevent presenting symptoms and signs [10]. Study Limitations First, the study sample is relatively small, and originates from a single referral center. Broader studies are, therefore, needed to establish the impact of an altered fibrinolytic profile on recovery of pulmonary perfusion after acute PE. Second, the fibrinolytic profile was assessed after the thromboembolic event had occurred, so that we cannot firmly establish a causal relationship between fibrinolysis impairment and persistent perfusion abnormalities in the lung. Third, the estimation of the time interval between symptoms onset and PE diagnosis cannot be absolutely precise for the perception of symptoms may vary substantially from one patient to another. Conclusion In conclusion, our data indicate that prolonged time between symptoms onset and PE diagnosis and fibrinolytic imbalance are both independent predictors of incomplete recovery of pulmonary perfusion after acute PE. Authors Contribution Conception and design: DL, APC, RA, DP. Collection and analysis of data: DL, APC, CF, MB, MM. Statistical analysis: DL. Drafting the manuscript: DL, APC, MM. Critical review of the manuscript: EA, RM, LM, RA, DP. References [1] Stein PD, Yaekoub AY, Matta F, Janjua M, Patel RM, Goodman LR, et al. Resolution of pulmonary embolism on CT pulmonary angiography. Am J Roentgenol 2010; 194:1263–8. [2] Cosmi B, Nijkeuter M, Valentino M, Huisman MV, Barozzi L, Palareti G. Residual emboli on lung perfusion scan or multidetector computed tomography after a first episode of acute pulmonary embolism. Intern Emerg Med 2011;6:521–8. [3] Alonso-Martínez JL, Anniccherico-Sánchez FJ, Urbieta-Echezarreta MA, GarcíaSanchotena JL, Herrero HG. Residual pulmonary thromboemboli after acute pulmonary embolism. Eur J Intern Med 2012;23:379–83. [4] Golpe R, de Llano LA, Castro-Añón O, Vázquez-Caruncho M, González-Juanatey C, Fariñas MC. Long-term outcome of patients with persistent vascular obstruction on computed tomography pulmonary angiography 6 months after acute pulmonary embolism. Acta Radiol 2012;53:728–31. [5] Lisman T, de Groot PG, Meijers JCM, Rosendaal FR. Reduced plasma fibrinolytic potential is a risk factor for venous thrombosis. Blood 2005;105:1102–5. [6] Guimarães AH, de Bruijne EL, Lisman T, Dippel DW, Deckers JW, Poldermans D, et al. Hypofibrinolysis is a risk factor for arterial thrombosis at young age. Br J Haematol 2009;145:115–20.
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[7] Undas A, Cieśla-Dul M, Drążkiewicz T, Sadowski J. Altered fibrin clot properties are associated with residual vein obstruction: effects of lipoprotein(a) and apolipoprotein(a) isoform. Thromb Res 2012;130:184–7. [8] Cellai AP, Lami D, Antonucci E, Fiorillo C, Becatti M, Olimpieri B, et al. Fibrinolytic inhibitors and fibrin characteristics determine a hypofibrinolytic state in patients with pulmonary embolism. Thromb Haemost 2013;109:565–7. [9] Miniati M, Fiorillo C, Becatti M, Monti S, Bottai M, Marini C, et al. Fibrin resistance to lysis in patients with pulmonary hypertension other than thromboembolic. Am J Respir Crit Care Med 2010;181:992–6. [10] Miniati M, Cenci C, Monti S, Poli D. Clinical presentation of acute pulmonary embolism. A survey of 800 cases. PLoS ONE 2012;7:e30891. [11] Miniati M, Pistolesi M, Marini C, Di Ricco G, Formichi B, Prediletto R, et al. Value of perfusion lung scan in the diagnosis of pulmonary embolism: results of the Prospective Investigative Study of Acute Pulmonary Embolism Diagnosis (PISA-PED). Am J Respir Crit Care Med 1996;154:1387–93. [12] Meyer G, Collignon MA, Guinet F, Jeffrey AA, Barritault L, Sors H. Comparison of perfusion lung scanning and angiography in the estimation of vascular obstruction in acute pulmonary embolism. Eur J Nucl Med 1990;17:315–9. [13] Simonneau G, Sors H, Charbonnier B, Page Y, Laaban JP, Azarian R, The THESEE Study Group, et al. A comparison of low molecular weight heparin with unfractionated heparin for acute pulmonary embolism. N Engl J Med 1997;337:663–9. [14] Wartski M, Collignon MA, for the THESEE Study Group. Incomplete recovery of lung perfusion after 3 months in patients with acute pulmonary embolism treated with antithrombotic agents. J Nucl Med 2000;41:1043–8. [15] Miniati M, Monti S, Bottai M, Scoscia E, Bauleo C, Tonelli L, et al. Survival and restoration of pulmonary perfusion in a long-term follow-up of patients after acute pulmonary embolism. Medicine (Baltimore) 2006;85:253–62. [16] Miniati M, Monti S, Airò E, Pancani R, Bauleo C, Formichi B, et al. Accuracy of chest radiography in predicting pulmonary hypertension. Thromb Res 2014;133:345–51. [17] Galiè N, Hoeper MM, Humbert M, Torbicki A, Vachiery JL, Barbera JA, et al. The Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS), endorsed by the International Society of Heart and Lung Transplantation (ISHLT) Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Heart J 2009; 30:2495–537. [18] Carter AM, Cymbalista CM, Spector TD, Grant PJ, Heritability of Clot Formation, Morphology, and Lysis: The EuroCLOT Study, on behalf of the Euro CLOT investigators. Arterioscler Thromb Vasc Biol 2007;27:2783–9. [19] Cellai AP, Lami D, Magi A, Liotta AA, Rogolino A, Antonucci E, et al. Assessment of fibrinolytic activity by measuring the lysis time of a tissue-factor-induced clot: a feasibility evaluation. Clin Appl Thromb Hemost 2010;16:337–44. [20] Morris TA, Marsh JJ, Chiles PG, Auger WR, Fedullo PF, Woods Jr VL. Fibrin derived from patients with chronic thromboembolic pulmonary hypertension is resistant to lysis. Am J Respir Crit Care Med 2006;173:1270–5. [21] Palka I, Nessler J, Nessler B, Piwowarska W, Tracz W, Undas A. Altered fibrin clot properties in patients with chronic heart failure and sinus rhythm: a novel prothrombotic mechanism. Heart 2010;96:1114–8. [22] Mills JD, Ariëns RA, Mansfield MW, Grant PJ. Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. Circulation 2002;106:1938–42. [23] Meltzer ME, Lisman T, de Groot PG, Meijers JC, le Cessie S, Doggen CJ, et al. Venous thrombosis risk associated with plasma hypofibrinolysis is explained by elevated plasma levels of TAFI and PAI-1. Blood 2010;116:113–21. [24] Lang IM, Marsh JJ, Olman MA, Moser KM, Loskutoff DJ, Schleef RR. Expression of type 1 plasminogen activator inhibitor in chronic pulmonary thromboemboli. Circulation 1994;89:2715–21. [25] Undas A, Zawilska K, Ciesla-Dul M, Lehmann-Kopydłowska A, Skubiszak A, Ciepłuch K, et al. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood 2009;114:4272–8. [26] Blombäck B. Fibrinogen and fibrin–proteins with complex roles in hemostasis and thrombosis. Thromb Res 1996;83:1–75. [27] Sanchez O, Helley D, Couchon S, Roux A, Delaval A, Trinquart L, et al. Perfusion defects after pulmonary embolism: risk factors and clinical significance. J Thromb Haemost 2010;8:1248–55.