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From the Departments of *Anesthesiology, and Surgery (Cardiothoracic), Emory University School of Medicine, Atlanta, Georgia.
Address correspondence and reprint requests to Kenichi A. Tanaka, MD, MSc, Division of Cardiothoracic Anesthesia, Department of Anesthesiology, 1364 Clifton Rd., NE, Atlanta, GA 30322. Address e-mail to kenichi.tanaka{at}emoryhealthcare.org.
Abstract
BACKGROUND: Antithrombin (AT) levels decrease during cardiopulmonary bypass (CPB), particularly when combined with deep hypothermic circulatory arrest (DHCA). Low AT levels might lead to imbalance of pro- and anticoagulant factors promoting systemic thrombotic events. We hypothesized that low levels of AT might lead to increased in vitro thrombin generation when procoagulant factors are added to the patient's plasma after CPB.
METHODS: Blood samples were obtained before heparinization and after separation from CPB from five patients undergoing cardiac surgery with DHCA. AT levels were determined by chromogenic assay and expressed as a percent of normal activity. The balance between procoagulant and anticoagulant elements was manipulated in the patients' plasma by adding normal donor plasma, AT-deficient plasma, or purified AT. The ThrombinoscopeTM system was used to evaluate thrombin generation with and without AT supplementation.
RESULTS: AT levels (median, range) were 82.0% (71.0, 109) and 37.0% (34.0, 41.0) of normal before and after separation from CPB, respectively (P < 0.05). Peak thrombin generation (median, range) was 56.6 nM (42.1, 61.0) in plasma after CPB, and it remained at 61.1 nM (54.9, 64.5) when a donor plasma with normal AT (105%) was added. When AT-deficient plasma was added to the patient's plasma, peak thrombin generation (median, range) was increased from 56.6 nM (42.0, 61.0) to 117 nM (95.0, 188) (P < 0.05 versus control). After the addition of purified AT, the peak thrombin generation was reduced to 12.2 nM (9.0, 29.3) (P < 0.05 versus control).
CONCLUSION: Plasma AT activity is severely decreased after CPB with DHCA. Our data suggest that the administration of coagulation factor components without AT repletion may lead to excessive thrombin generation, which clinically, may potentially lead to a hypercoagulable state.
Complex cardiac surgical procedures requiring prolonged cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) often lead to profound coagulopathy due to consumptive loss and hemodilution of procoagulant proteins. Although often overlooked by clinicians, there are concomitant decreases in anticoagulant protein activities as well.1–4 Catastrophic systemic thromboses have been reported after heparin reversal with protamine and after hemostatic blood product transfusion to patients after CPB during the antifibrinolytic therapy (Table 1).5–13 Collectively, these cases suggest that there is a delicate balance between pro- and anticoagulant factors and that this balance can be rapidly disrupted by interventions to establish hemostasis.
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Antithrombin (AT) is an anticoagulant protein that inhibits nonclot bound thrombin and activated factor X in plasma (Fig. 1). Its importance in regulating coagulation is demonstrated by embryonic lethality in AT knockout mice,14 as well as the association between low AT levels and venous thrombosis.15 AT activity levels decrease to about 50%–60% of normal during CPB of durations between 60 and 90 min,2,3 but may decrease even lower when DHCA is used.1,4 The greater decrease in AT activity related to DHCA is likely due to increased CPB duration and the greater complexity of surgical procedures performed.16 It seems possible that acute systemic thrombus formation in this situation could result from unregulated thrombin generation. In this in vitro study, we hypothesized that the low AT levels after CPB with DHCA would accentuate thrombin generation following hemostatic blood transfusion.
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METHODS
All procedures met the approval of the Emory University IRB and were performed after receiving written consent from the patients undergoing elective surgery with CPB and DHCA (n = 5). Inclusion criteria included age over 18 yr and normal prothrombin time (<14.9 s), platelet count (150–400 x 106/mL), and fibrinogen levels (250–450 mg/dL) before surgery. Patients with preexisting hepatic (elevated aspartate aminotransferase or alanine aminotransferase) or renal (serum creatinine 
1.5) dysfunction were excluded. In all patients, 400 U/kg heparin was given before institution of CPB with hourly supplements of 100 U/kg. Per our institutional policy for DHCA procedures, aprotinin (Trasylol, Bayer, West Haven, CT) was administered as a 2 million kallikrein inhibiting units (KIU) initial loading dose, followed by 2 million KIU in the CPB priming solution, and an infusion of 500,000 KIU/h. Transfusion of packed red blood cells during CPB was at the discretion of the attending anesthesiologist. No hemostatic blood products (i.e., fresh frozen plasma [FFP], cryoprecipitate, or platelets) were transfused before collection of blood samples after CPB.
Blood Sample Collection
Patient blood samples were obtained before heparin administration and after CPB at the completion of protamine infusion (250–300 mg). Whole blood samples were collected into glass tubes containing 3.2% buffered citrate and platelet poor plasma was obtained by centrifugation of the blood at 3000g for 15 min. Plasma AT activity was measured before and after CPB using the chromogenic assay (Coamatic Antithrombin kit, Diapharma Group, Inc., West Chester, OH).
Thrombin Generation in Platelet Poor Plasma
The balance between procoagulant and anticoagulant elements was manipulated by adding normal plasma, AT-deficient plasma, or purified AT to the plasma obtained from the patients after CPB. From a clinical perspective, each of these additions corresponds to the administration of FFP, procoagulant factor component(s) (e.g., factor VIIa), and AT concentrate, respectively. A stock of normal plasma was prepared from citrated whole blood obtained from a single healthy volunteer after informed, written consent. Its AT activity was 105% (normal range, 80%–120%). AT-deficient plasma was obtained from Enzyme Research Laboratories (South Bend, IN).
Thrombin activity was detected by changes in fluorescence intensity after cleavage of a fluorogenic substrate, Z-Gly-Gly-Arg-AMC (benzyloxycarbonyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, Bachem Bioscience, King of Prussia, PA) as previously described.3,17–19 The plasma was treated with 4 IU/mL of heparinase-I (IBEX, Montreal, Canada) because residual heparin effects might completely obtund thrombin generation in coagulopathic plasma. For thrombin generation measurements, 80 µL of platelet poor plasma followed by 20 µL of an activator (Biodis, Signes, France) was added to microtiter plate wells. The plate was incubated for 2–3 min at 37°C and then 20 µL of the substrate buffer was added. A thrombin calibrator with known thrombin-like activity was monitored in parallel sample wells to allow for calculation of generated thrombin in nM. The progress of the reaction was continuously monitored for 70 min at 37°C with a fluorescence reader (Fluoroscan Ascent, Thermo Labsystems, Franklin, MA) equipped with a 390 nm excitation filter and a 460 nm emission filter. A dedicated software program (Thrombinoscope, Synapse BV, Maastricht, The Netherlands) was used to record the peak thrombin level.
The schemata of the study are shown in Figure 2. The thrombin generation measurements were performed in aliquoted platelet-poor plasma samples (200 µL) from blood obtained after CPB as follows: Tube 1: plasma without any supplement; Tube 2: plasma supplemented with normal plasma (40 µL of the single donor plasma); Tube 3: plasma supplemented with AT-deficient plasma (40 µL of AT-deficient plasma); and Tube 4: plasma supplemented with purified human AT (4.7 µg). The volume of the added normal plasma, 40 µL, approximates 4 U of FFP transfusion (1000–1200 mL) given to a 70-kg patient. The AT supplementation was modeled by adding purified AT (23.5 µg/mL, final concentration) to the patient plasma. Assuming a normal plasma concentration of 140 µg/mL and an AT activity of about 40% in our post-CPB samples, the AT concentration was increased to an activity level of 50%–60% (
79.5 µg/mL).
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Statistical Analysis
Based on previous studies of thrombin generation, a sample size of 5 was needed to detect a 30% change from the controls in peak thrombin generation with a [β] 0.8 and an [
] < 0.05.3,19 The Kruskal–Wallis H-test followed by the Mann–Whitney U-test with Bonferroni's correction was used to compare the percentage changes in peak thrombin generation among samples using SPSS 15.0 (SPSS Inc., Chicago, IL). Data are expressed as the median (ranges). P 
0.05 was considered significant.
RESULTS
Demographics and operative data are presented in Table 2. Decreased procoagulant activity after CPB was evident from significant decreases in platelet count and fibrinogen levels, and a prolonged international normalized ratio of prothrombin time compared with baseline measurements. There was a concomitant decrease in the activity of a natural anticoagulant AT (<40% of baseline) after CPB compared with the measurements before CPB.
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Peak thrombin generation was 56.6 nM (42.1, 61.0) in the control plasma after CPB, and it remained at 61.1 nM (54.9, 64.5) (P = 0.9 versus control) when normal donor plasma was added to the control. When AT-deficient plasma was added to the patients' plasma, the peak thrombin generation was increased from 56.6 nM to 117 nM (95.0, 188) (P < 0.05 versus control). After the addition of purified AT, the peak thrombin generation was reduced to 12.2 nM (9.0, 29.3) (P < 0.05 versus control). The representative tracings of in vitro thrombin generation are shown in Figure 3.
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DISCUSSION
In this study, we demonstrate that prolonged CPB with DHCA results in severe decreases in AT to levels lower than previously reported after CPB.1–4 Our in vitro data illustrate the delicate balance between hemostatic and anticoagulant elements in patients undergoing prolonged CPB.20,21 When AT-depleted plasma was added to the patients' plasma obtained after CPB, peak thrombin generation was higher than when normal donor plasma with normal AT activity was added to these same samples. Conversely, when purified AT was added to the patients' plasma obtained after CPB, peak thrombin generation was reduced compared with baseline.
During normal hemostasis, thrombin is largely bound to the local thrombus at the site of a vascular injury (Fig. 1). A small amount of thrombin may "leak" out of the thrombus, but AT efficiently inhibits thrombin that would otherwise propagate thrombus formation via positive feedback activations of factors V, VIII, and XI.17 In fact, AT normally exists in plasma at a much higher concentration (2.4 µM) than the concentration of thrombin (0.36 µM) generated in thrombus.22 Decreases in coagulation factors, platelets, and fibrinogen, as may be observed after CPB, reduce thrombin generation and fibrin formation at the site of vascular injury (Fig. 3) increasing the risk for microvascular bleeding.23,24 The treatment of postoperative bleeding is often focused on supplementing hemostatic elements including procoagulant factors, platelet concentrate, and fibrinogen-rich cryoprecipitate.4,21 These procoagulant interventions may help establish local hemostasis at vascular injury sites, but they may not restore normal levels of anticoagulant factors such as AT. Thus, systemic thromboses may potentially be triggered when AT fails to inhibit nonthrombus bound thrombin (Fig. 1). Indeed, in this experiment, the addition of volunteer plasma with normal AT activity did not change thrombin generation compared with the nonsupplemented control experiments. Furthermore, because antifibrinolytic drugs are commonly used for complex cardiac procedures (Table 1), the breakdown of intravascular thrombus via endogenous fibrinolytic enzymes may be inhibited.25 Consequently, the balance of coagulation versus fibrinolysis may favor intravascular thrombosis when natural anticoagulant elements such as AT are iatrogenically reduced by prolonged CPB.
AT activity may not be sufficiently restored after the transfusion of blood products (FFP, platelet concentrate, or cryoprecipitate) after CPB. FFP contains 200–240 U of AT in 250–300 mL of volume. Thus, multiple units of plasma need to be transfused to achieve the equivalent AT as achieved from a single dose of AT concentrate (624 unit per vial). Despite the cost of AT ($985 per AT vial versus $75 per unit of FFP), the use of AT concentrate may be preferable for AT repletion than FFP because the latter increases the risk of excessive intravascular volume and the risk associated with allogeneic blood transfusion complications including transfusion- related acute lung injury.26,27
Our results need to be interpreted with caution. In vitro coagulation tests in plasma do not take into consideration the influence of blood flow, monocytes, platelets, and vascular endothelial interactions on overall coagulation processes.28,29 Additionally, the optimal AT level after CPB has not been determined. There may be increased postoperative bleeding when AT is restored to its normal physiological level,2 while AT levels <58% of normal have been shown to be associated with both acute and subacute adverse events, including mediastinal reexploration, prolonged hospitalization, and thromboembolic complications.30 We therefore believe a large prospective study is warranted to investigate strategies to safely use AT concentrates, coagulant factors, and antifibrinolytic drugs to achieve balanced hemostasis and improve outcomes in patients undergoing complex cardiac surgical procedures.
Footnotes
Accepted for publication October 26, 2007.
Dr. Jerrold H. Levy, Section Editor for Hemostasis and Transfusion Medicine, was recused from all editorial decisions related to this manuscript.
REFERENCES
This article has been cited by other articles:
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  D. Bolliger, F. Szlam, R. J. Molinaro, N. Rahe-Meyer, J. H. Levy, and K. A. Tanaka Finding the optimal concentration range for fibrinogen replacement after severe haemodilution: an in vitro model Br. J. Anaesth., June 1, 2009; 102(6): 793 - 799. [Abstract] [Full Text] [PDF]
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 K. A. Tanaka, N. S. Key, and J. H. Levy Blood Coagulation: Hemostasis and Thrombin Regulation Anesth. Analg., May 1, 2009; 108(5): 1433 - 1446. [Abstract] [Full Text] [PDF]
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