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J Thorac Cardiovasc Surg 2003;125:336-343
© 2003 The American Association for Thoracic Surgery
Surgery for Acquired Cardiovascular Disease (ACD) |
From the Department of Cardiac Surgery, Centro Cardiologico Monzino IRCCS,a Department of Pharmacological Sciences, E. Grossi Paoletti Center, University of Milan,b and Department of Biostatistics, European Institute of Oncology,c Milan, Italy.
This work was supported in part by a grant from the Italian Ministry of Health (ICS 030.6 RF99.14).
Received for publication Feb 14, 2002. Revisions requested June 13, 2002; revisions received June 27, 2002. Accepted for publication July 15, 2002. Address for reprints: Alessandro Parolari, MD, PhD, Department of Cardiac Surgery, University of Milan, Centro Cardiologico, Fondazione Monzino IRCCS, Via Parea, 4, 20138, Milan, Italy (E-mail: alessandro.parolari{at}cardiologicomonzino.it).
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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1,2 Moreover, activation of both the fibrinolytic and inflammatory pathways can contribute to the so-called postpump syndrome, which is characterized by an impairment of myocardial, cerebral, pulmonary, and renal function. 3
Early activation of coagulation, as shown by the increased levels of prothrombin fragment F1.2 and thrombin-antithrombin complex (TAT), has been documented in patients who underwent CPB during cardiac surgery. 4-6 Activation of the fibrinolytic system follows. Indeed, increased levels of D-dimer (XDP) 7 and plasmin-antiplasmin complex 4 have been reported as a consequence of the activation of the coagulation system.
Activation of coagulation recognizes multiple pathways that are related to various aspects of the intervention, such as surgical trauma, interaction of blood with extensive nonendothelial surfaces of the bypass circuit, and retransfusion of pericardial blood. 6,8 More recently, tissue factor (TF) expressed by monocytes during and immediately after the procedure has been reported to play a pivotal role in CPB-induced coagulopathy. 6,9 The end point of both intrinsic and extrinsic coagulation pathways is indeed the generation of thrombin, the prominent enzyme in the activation of hemostasis.
The behavior of hemostatic variables immediately after and in the time frame that spans from a few hours to 2 to 3 days after cardiac surgery with CPB is widely documented. 8 On the contrary, less information is available about the behavior of these variables in a longer follow-up period. The lack of information is due to the fact that patients with uncomplicated procedures are usually discharged from the hospital 4 to 8 days after the operation, making it difficult to recall them over time.
Previous studies from our group and others 10-12 have reported increased thrombin generation and alteration of fibrinolytic activity in patients undergoing CPB that lasted up to 8 to 30 days after the intervention and that were not prevented by the administration of aprotinin during the procedure. 10
On this basis, it might be hypothesized that cardiac surgery with CPB activation of the coagulation-fibrinolytic pathways occurs not only during the operation but also later. The goal of the present study was therefore to assess whether alterations of the coagulation persist for longer follow-up periods. Moreover, the contribution of TF from mononuclear leukocytes in the occurrence of activation of coagulation within the first week after the intervention has been investigated.
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Patients and methods |
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13 Preoperative ejection fraction was greater than 30%, and left ventricular end-diastolic pressure was less than 20 mm Hg. In order to avoid excessive blood sampling from the same subject, coagulation/fibrinolytic variables were measured in 16 patients whereas blood from 10 patients was processed for peripheral blood mononuclear leukocyte (PBML) isolation. All patients provided informed consent to participate in this study that was approved by the Ethical Committee of Centro Cardiologico Monzino IRCCS. None of the patients had renal or liver dysfunction or were taking drugs affecting coagulation-fibrinolytic variables.
Surgical procedure
All patients underwent intervention by the same surgical team. On the morning of the operation, patients received their usual dose of antianginal drugs, with 2 to 5 mg of morphine and 1 mg of atropine as premedication. Anesthesia was induced by the administration of sodium thiopental (3 mg/kg), fentanyl (0.75 µg/kg), succinylcholine (1 mg/kg), diazepam (10 mg), and pancuronium bromide (0.1 mg/kg). After endotracheal intubation, patients were ventilated to normocapnia with an oxygen and air mixture. Boluses of fentanyl (with or without droperidol), diazepam, and pancuronium bromide were given when necessary. Cefuroxime (2 g) was given intravenously for infection prophylaxis. A radial artery catheter and a flow-directed pulmonary artery catheter (Swan-Ganz, Baxter/American Edwards Laboratories) were inserted for hemodynamic measurements. The extracorporeal circuit consisted of a roller pump, hollow-fiber oxygenator with integrated heat exchanger, arterial filter, open cardiotomy reservoir, and polyvinyl tubing system. The extracorporeal circuit was primed with 1500 mL of crystalloid solution and 5000 IU of bovine lung heparin. After systemic heparinization (300 IU/kg bovine lung heparin), CPB was initiated by the placement of cannulas in the ascending aorta and right atrium (2-stage venous cannula). Activated clotting time was kept at 440 seconds or greater with additional heparin. The nonpulsatile flow rate was maintained at 2.4 Lxmin-1xm-2 during the cooling and rewarming phases and at 2.0 Lxmin-1xm-2 during stable hypothermia. Before and after CPB, blood from mediastinum was collected with a cell-saver device, washed, and returned to the patient, whereas during CPB, mediastinal blood was returned directly to the extracorporeal circuit. Mean arterial pressure was kept at 50 to 90 mm Hg. Patients were cooled to 29°C to 31°C and received a first dose (1000 mL) of cool (4°C antegrade and retrograde high-potassium crystalloid cardioplegia (St Thomas Hospital II Cardioplegic Solution, SALF) just after aortic crossclamping. This maneuver was repeated (250 mL retrogradely) every 20 minutes of aortic crossclamp time. Disturbances in the acid-base balance were appropriately treated, and acid-base equilibrium was maintained by using the alpha-stat method. The hematocrit level was maintained within 18% to 25% throughout the procedure.
After termination of the intervention, heparin was antagonized with protamine sulfate at a 1:1 ratio (3 mg/kg). None of the patients required inotropic support for weaning from CPB. After the operation, patients were admitted to the intensive care unit (ICU) and treated according to a standardized protocol. Mean arterial blood pressure was kept at 70 to 90 mm Hg, heart rate at 70 to 90 beats/min, and cardiac index at greater than 2.0 Lxmin-1xm-2. Patients were ventilated to normocapnia, and an arterial oxygen tension of 80 mm Hg with continuous positive-pressure ventilation and positive end-expiratory pressure of 5 cm H2O was maintained until extubation, according to the ICU regimen. Basic fluid administration consisted of 0.9% NaCl and polygelatine. Packed erythrocytes were infused when the hematocrit level was less than 18% during CPB and less than 24% in the ICU. When the cardiorespiratory condition had stabilized, patients were transported to the ward for further recovery. In the postoperative period, patients received subcutaneous heparin (5000 IU twice daily), starting from 24 hours to 72 hours after CPB, followed by aspirin (325 mg/day), which was usually given within the first and second days after the intervention.
Follow-up
All the patients were hospitalized until the eighth postoperative day. Sixteen of them had periodic follow-up visits (physical examination, electrocardiography, and blood collection for coagulation-fibrinolytic variables) at the 15th postoperative day and every 15 days on completion of the study protocol at the 60th postoperative day.
Blood sampling
Blood collection was performed from a peripheral vein through a 19-gauge needle in plastic tubes (0.13 mol/L sodium citrate or 15 U/mL heparin as an anticoagulant, see below) during the morning. Blood for measurement of coagulation-fibrinolytic variables was collected at the following time points: the day before the operation (-1) and the 4th (+4), 8th (+8), 15th (+15), 30th (+30), 45th (+45), and 60th (+60) postoperative day. Blood for mononuclear leukocyte isolation was collected the day before the operation (-1) and at the fourth (+4) and eighth (+8) postoperative day.
Laboratory methods
For selected coagulation-fibrinolytic variable measurements, plasma was prepared by means of centrifugation at 1500g for 20 minutes at 4°C from blood collected in 0.13 mol/L sodium citrate (1:10 vol/vol) within 1 hour after venipuncture. Centrifugation was carried out at room temperature for factor VII determination. Plasma was divided into aliquots and frozen at -80°C until assay.
Fibrinogen levels were measured according to the method of Clauss 14 (Dade-Thrombin, Dade Diagnostics) with a coagulometer (Schnitger Gross).
TAT, XDP, and factor VII antigen levels were determined by using ELISA with commercially available kits, according to the manufacturers' recommendations (Enzygnost TAT, Dade Behring; Dimer-test Gold Stripwell EIA kit, Instrumentation Laboratory; and Asserachrom VII:ag, Stago Boehringer). Prothrombin fragment F1.2 was assayed in heparinized plasma by using a commercially available ELISA kit (Enzygnost F1.2, Dade Behring). Plasma factor VII coagulant was assessed by using a 1-stage clotting time assay with a coagulometer, with factor VII-deficient plasma and human tissue thromboplastin as reagents (Instrumentation Laboratory). Plasma factor VII activity was measured by using a 1-stage clotting assay with a soluble mutant recombinant TF (kind gift from J. H. Morrissey, University of Illinois) that possesses cofactor activity for factor VII activity but fails to support activation of factor VII. 15 Clotting times were converted to factor VII activity concentration (in nanograms per milliliter) by means of comparison with a standard curve of purified recombinant factor VII activity (National Institute of Biological Standards and Control, United Kingdom, code 89/688). Coagulation-fibrinolytic data were normalized for hematocrit values.
Blood sampling for PBML isolation
Blood was drawn in sterile tubes containing heparin (10 U/mL) and divided into 2 aliquots under sterile conditions, according to the method of Osterud and Bjorklid. 16 Saline solution or 2 ng/mL bacterial lipopolysaccharide (LPS; Sigma) was added, and samples were incubated at 37°C. Reaction was stopped after 2 hours by adding ethylenediamine tetra-acetic acid (2%, pH 7.4). Platelet-rich plasma was separated (2300 rpm for 4 minutes) and replaced with culture medium RPMI (BioWhittaker Italia) containing sodium citrate. PBMLs were obtained by means of sedimentation on Ficoll-Paque (Pharmacia) density gradient. Cells were washed, resuspended in PBS, and diluted to 2 x 106/mL. Aliquots (100 µL) of cell suspensions were subjected to repeated cycles of freezing and thawing and used to assess procoagulant activity.
Assay of procoagulant activity
Procoagulant activity was measured on cell lysates by using the plasma recalcification time assay. In brief, citrated plasma (100 µL) was incubated with cell lysate (100 µL), and 25 mol/L CaCl2 was added to initiate the reaction. Clotting times were converted into arbitrary units by using reference curves determined with a standard human placental thromboplastin preparation (Thromborel Behring). The logarithm of the clotting times was related to the logarithm of the procoagulant activity. Experiments performed with factor VII-deficient plasma verified that the procoagulant activity measured was TF activity. Data are therefore expressed as units of TF activity per milliliter, and the TF activity ratio of LPS-stimulated/LPS-unstimulated cells is reported.
Statistical analysis
Clinical data of patients are reported as means ± 1 SD or as percentages. Trends by time in prothrombin fragment F1.2, TAT, fibrinogen, XDP, factor VII (as antigen or in its coagulant and activated forms), and TF activity were studied by using a repeated measurements analysis, taking into account the correlation among the measurements at the different time periods. A variance component correlation structure was used, and there was no evidence that a more complex structure was needed. Contrasts were calculated to estimate the mean change of the different variables from baseline. The normality assumptions were checked by using normal plots, and logarithm transformation was used to achieve normality for prothrombin fragment F1.2, TAT, and XDP. One subject was excluded from the analysis because of TAT and prothrombin fragment F1.2 preoperative values exceeding 2 SDs.
Fractional polynomials were fitted to find suitable models for the curved relationship between the different variables and time. 17 In the graphs the predicted values from these models, as well as the observed values for each subject by time, are plotted. All calculations were performed by using PROC MIXED and PROC GLM in SAS software (SAS for Windows version 6.11, SAS Institute Inc).
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Results |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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Prothrombin fragment F1.2
Prothrombin fragment F1.2 levels increased significantly from baseline (0.35 ± 0.216 nmol/L) at the fourth and eighth day postoperatively. The increase from baseline values, as estimated from percentage changes on the least-squared means obtained from repeated measurements analysis, was 95.3% (95% confidence interval [CI], 38.5-175.5) and 57.4% (95% CI, 12.2-120.7) at the fourth and eighth day, respectively. Thereafter, these levels decreased, with the estimated percentage change being 27.8% greater than the baseline value 15 days after the intervention, with a complete return to baseline values occurring at the 45th postoperative day (Figure 1).
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TF activity
Before the operation, freshly isolated PBMLs expressed 1.44 ± 0.84 U/mL TF activity, and this increased to 5.01 ± 5.8 U/mL on exposure to 2 ng/mL LPS. TF activity in unstimulated cells did not change postoperatively, whereas it increased up to 8.05 ± 6.9 U/mL at the fourth postoperative day. TF activity expressed as the ratio of LPS-stimulated/LPS-unstimulated PBMLs showed a trend (P = .06) toward a significant increase at the fourth postoperative day respective to preoperative values (Figure 5).
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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18 and this value can increase further 3 months after the operation. 19 In addition to the problem of graft occlusions, other complications on a thrombotic basis can frequently occur. The incidence rates reported for perioperative stroke are usually from 2% to 3%, 20 reaching 5% in some reports. 21 The majority of strokes occur, however, in a time frame up to 10 days after surgical intervention, after awakening from surgical intervention, or after an uneventful neurologic recovery. 21 This observation suggests that factors other than those related to the complexity of the intervention might contribute to the occurrence of thrombotic events.
Our results indicate that alterations of the coagulation-fibrinolytic profile, which are markedly evident within 4 to 8 days from the intervention, persist for at least 2 months of the follow-up period. In more detail, fibrinogen levels were significantly higher at the 15th postoperative day, and TAT and XDP levels remained higher at the 30th and 60th postoperative days, respectively. This latter finding suggests that 2 months after the operation, the generation of plasmin still persists, both as a consequence of ongoing removal of hemostatic plugs and formation of uncovered intravascular fibrin deposition. D-dimer is in fact recognized as an indicator of fibrin formation in vivo. 22 Moreover, as suggested by others, 23 increased levels of XDP might result from a remodeling of clot in the operative site.
Up to now, only one study has evaluated the hemostatic-prothrombotic profile after CPB during a relative long-term follow-up (3 months). 12 In this study TAT and fibrinogen levels that were found to be significantly increased at the eighth day normalized after 3 months. Results from our study document that thrombin generation is still increased 2 months after the operation.
A trend toward increase of TF activity by PBMLs was detected 4 days postoperatively. This increase, regardless of a large interindividual variability, indicates that monocyte activation in terms of TF activity likely persists for at least some days after the operation.
The extracorporeal circuit has been previously shown to be critical in terms of monocyte activation. Indeed, Barstad and colleagues 9 showed an increase of TF in adherent monocytes retrieved from the oxygenators immediately after bypass arrest that was reduced when heparin-coated circuits and systemic heparinization were adopted. Moreover, as far as PBMLs are considered, alterations of the extrinsic pathway of coagulation have been documented during and soon after cardiac surgery performed with the use of CPB. 5,24 Ernofsson and associates 24 reported that TF expression was not changed at the end of CPB, whereas a significant increase of TF was recorded 20 hours after the operation. Interestingly, the same authors documented such an increase both in unstimulated monocytes and in monocytes exposed to LPS. These data allowed the conclusion that TF expression in circulating monocytes is upregulated the day after heart operation. Our results extend this observation and suggest that relatively increased levels of TF activity in the systemic circulation might persist longer after the operation, thus addressing a role of TF as a generator of thrombin not only during and immediately after the intervention but also in a more prolonged follow-up period.
Previous data by Nieuwland and coworkers 25 showed the presence of higher levels of procoagulant cell-derived microparticles in pericardial blood from patients undergoing CPB with respect to systemic circulation. The finding about an increased procoagulant profile of the blood collected from the pericardial cavity respective to systemic circulation was also confirmed by a more recent study. 26 These results, however, have been obtained either after the release of the aortic clamp (from 5-10 minutes) or in a time frame from 15 to 60 minutes on bypass, respectively. In the latter condition, plasma TF from the systemic circulation was not increased, thus reflecting a different behavior between cells involved in the surgical damage and those from peripheral blood 26 under the profile of time requested for TF to be exposed.
The observed upregulation of TF activity is strictly paralleled by activation of coagulation detected as an increase of prothrombin fragment F1.2 levels used as a marker for thrombin generation. The coagulopathy profile persists for longer time periods from the intervention, as documented by fibrinogen and TAT levels that are still increased 15 and 30 days postoperatively, respectively.
One limitation of this study is that no control group of patients undergoing another type of operation was studied; for this reason, this study cannot clearly address whether changes in coagulation-fibrinolytic variables are specific for cardiac surgery with CPB or common to all surgical procedures and wound healing. This issue is rather controversial. Indeed, it has been previously shown that coagulation and fibrinolytic variables are altered after some types of skeletal surgery 27 and, in contrast, that a major activation of coagulation and fibrinolysis occurs after cardiac surgery performed with CPB but not after general thoracic surgery. 7 Regardless of the different conclusions, it should be pointed out that the observation period considered in the above studies is limited to the first 24 to 48 hours after the operation. No information, however, is available about the behavior of the considered variables for longer periods of time.
At present, current indication for the prevention of cardiovascular events in patients undergoing CPB is to prescribe aspirin. 18 Our observation of a persistent activation of coagulation 2 months after CPB raises the question of whether antithrombotic therapy should be reconsidered. However, this question needs to be answered on the basis of the results emerging from appropriately designed clinical trials.
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