Antithrombin (AT) is a serine protease inhibitor (serpin) and the principal inhibitor of the final common pathway of the coagulation system by inactivation of circulating thrombin (factor IIa) and factor Xa, among other serine proteases. Heparin increases AT activity 2000- to 4000-fold due to a conformational change in the quaternary structure of AT by heparin binding and through formation of a ternary complex of thrombin, AT, and heparin. Heparin-augmented AT activity is still the principal mechanism of anticoagulation for cardiopulmonary bypass (CPB).
AT levels are decreased after administration of heparin due to degradation of the ternary complex. Additionally, acquired AT deficiency is common in patients with critical illness, severe hepatic dysfunction, and after major cardiovascular surgery.1,2 The magnitude of reduction in AT after cardiac surgery is similar to that in patients with heterozygous AT deficiency, which is associated with increased risk of thromboembolic events.3,4 After cardiac surgery, lower levels of AT have been independently associated with prolonged intensive care unit (ICU) stay and a higher incidence of neurologic and thromboembolic events.5 We therefore examined for independent association between perioperative AT activity and the frequency of postoperative major adverse cardiac events (MACEs) in patients undergoing coronary artery bypass graft (CABG) surgery.
The study cohort was obtained from a continuing prospective longitudinal parent study of 1447 patients undergoing primary CABG surgery with CPB between August 2001 and May 2006 at 2 United States academic medical centers (CABG Genomics Program; http://clinicaltrials.gov/show/NCT00281164). With IRB approval, written informed consent was obtained from each patient. Patients were excluded from the parent study if they were younger than 20 yr old, underwent repeat or off-pump CABG, had a preoperative hematocrit <25%, or if they had received leukocyte-rich blood products within 30 days before surgery. All patients enrolled in the parent study were included. Patients without postoperative cardiac troponin I (cTnI) levels (n = 43) were excluded from further analysis. Demographic data, medical and surgical history, medications, and outcomes were recorded by trained research staff using defined protocols in a purpose-built case report form. The examination of the relationship between AT levels and MACE was not prespecified in the original parent study.
Perioperative anticoagulation protocols differed between institutions. At Brigham and Women's Hospital, patients received 300 U per kg body weight of porcine heparin to achieve an activated clotting time (ACT) of >400 s, until February 2004. From February 2004, patients received a Hepcon HMS Plus (Medtronic, Minneapolis, MN) calculated dose of porcine heparin to achieve an ACT of either 300 or 350 s. At Texas Heart Institute, 300 U per kg body weight of either bovine or porcine heparin was given to achieve an ACT of >400 s.
Clinical End Points for the Test Cohort
The primary clinical end point was prespecified as the occurrence of a MACE, defined as a composite outcome of any one or more of the following: postoperative mortality (defined as all deaths occurring within 30 days of the operation or occurring during the primary hospitalization), reoperation for coronary graft occlusion, myocardial infarction (MI) (predefined as peak postoperative cTnI concentration >12 ng/mL, being the upper 8th percentile), cardiac arrest (defined as a postoperative event requiring cardiopulmonary resuscitation) until first hospital discharge, thromboembolic event consisting of stroke (defined as a clinical diagnosis of focal or global neurological deficit), or pulmonary embolism (diagnosed by ventilation perfusion scan of moderate to high probability or by a positive pulmonary angiogram).
Cardiac Biomarker Assay
Blood samples were obtained before surgery, 5 min after administration of post-CPB protamine, and on the mornings of postoperative days (PODs) 1–5. Citrated plasma was stored in vapor-phase liquid nitrogen until analysis for cTnI with a sandwich immunoassay on a Triage® platform using monoclonal and polyclonal antibodies (Biosite, San Diego, CA) at a single core facility. Patient caregivers were not aware of the results of the assays as they were performed after patient discharge.
AT activity was measured with a colorimetric method using a Modular Analytics biochemistry analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY). The assay limit of quantitation was 21.6%. To report human plasma AT activity results in IU/mL, the National Institute for Biological Standards and Control Second International Reference Standard was used to determine a conversion factor of activity in IU/mL = activity in %× 0.0102. AT content was measured using an immunoephelometric method using a BN-100 ProSpec nephelometer (Dade Behring Diagnostics, Marburg, Germany). The assay limit of quantitation was 0.00672 mg/mL. To report human plasma AT content results in IU/mL, the National Institute for Biological Standards and Control Second International Reference Standard was used to determine a conversion factor of content in IU/mL = content in g/L × 3.64. Both assays measure free AT rather than AT complexed with heparin. Assays were performed by Charles River Laboratory in Montreal, Canada by personnel blinded to outcome status. Subsequent comparison of paired AT activity and content data revealed high correlation (r2 = 0.878), so only the activity is reported.
Statistical analyses were performed using SAS, version 9.1.3, and JMP 7.0 (SAS Institute, Cary, NC). AT activity was normally distributed at all time points, so was not transformed. Data are presented as mean (SD) and median with 10%–90% interquantile range, unless otherwise stated. Continuous variables were compared using analysis of variance or Wilcoxon Mann-Whitney ranked sum test when appropriate. Categorical variables were compared with χ2 or Fisher's exact test.
Multivariate logistic regression modeling was performed to identify and account for MACE risk factors that might confound any association between low AT activity and MACE. The multivariate analysis used a forward stepwise technique to identify independent risk factors for MACE, whereby clinically relevant demographic variables and variables with a two-tailed univariate P ≤ 0.2 were entered into the model and P ≤ 0.2 was necessary to remain in the model. Age, gender, race, body mass index, and institution were forced into the model. Nagelkerke generalized r2 and likelihood ratio test were used to determine the additional predictive value of AT upon MACE. F tests were used to compare generalized r2. Odds ratios and 95% confidence intervals for a 0.1 IU/mL decrease in AT activity were estimated. A two-sided P < 0.05 was considered significant.
The cohort comprised 1403 patients undergoing CABG surgery whose characteristics are described in Table 1. MACE occurred in 146 patients (10.4%), consisting of postoperative mortality (n = 12), MI (n = 108), stroke (n = 17), pulmonary embolism (n = 8), cardiac arrest (n = 16), or a subsequent postoperative or catheter-based treatment for graft occlusion (n = 6). Nineteen patients had 2 or 3 events, usually MI, with either subsequent death or stroke. Most adverse events occurred before or on POD 2. Of 12 patients with operative mortality, 2 patients died at POD 0 and 10 patients died on or after POD 5. Of 17 patients with stroke, 6 patients had a stroke on or before POD 2. Of 108 patients with MI, 89 patients had a diagnosis of MI first occurring on POD 1. MACE frequency did not differ between institutions (Table 1).
AT activity and content were measured at the 7 time points (Table 2). AT activity at baseline did not differ between patients with MACE (0.91 ± 0.13 IU/mL; n = 146) and those without MACE (0.92 ± 0.13 IU/mL; n = 1257) (P = 0.18). AT activity was significantly reduced at the post-CPB measurement compared with the preoperative time point (P < 0.0001) and returned to baseline levels over the ensuing 5-day period in patients with and without MACE. Postoperative AT activity was significantly lower in patients with MACE than those without MACE (Table 2).
Decreased preoperative AT activity was independently predicted by older age, male gender, and prior heparin use within the same hospitalization (Table 3). Other clinical variables that may possibly be indicative of recent heparin use at prior recent hospitalization, such as recent MI, were also independently predictive. Decreased preoperative AT activity was also independently associated with lower platelet count and increased partial thromboplastin time, independent of recent heparin use, perhaps indicating a dose effect of heparin administration upon decreased AT activity. In the 1205 patients who had complete data for all variables in the model, decreased post-CPB AT activity was independently predicted by lower preoperative AT activity and clinical variables that indicate greater hemodilution, such as lower body weight and height and increased transfusion incidence or a prolonged procedure (Table 4).
A clinical model predicting MACE was developed (Table 5; adjusted r2 = 0.156) for 1403 patients who had complete data for all variables in the clinical model. Variables that have been associated with MACE in prior studies, notably recent MI, longer perfusion time, a requirement for intraaortic counterpulsation, and red blood cell transfusion were also associated with MACE in this study. AT activities at baseline, post-CPB, PODs 1–5, and the change from baseline at these time points for each patient were added to the clinical model, one time point at a time. Preoperative, post-CPB, and POD 1 AT activity were not independently predictive of MACE, whereas AT activity on PODs 2 and 3 was independently predictive of MACE. MACE was independently associated with change in AT activity from baseline on PODs 2–5 (Table 5). The receiver operating characteristic of the model was significantly improved by the addition of AT activity on PODs 2–4 to the model (Fig. 1).
This prospective, observational, cohort study confirms earlier findings that preoperative AT activity is reduced in patients with recent exposure to heparin or recent MI.5,6 Furthermore, AT activity is reduced after CPB, likely due to consumption by heparin administration6 and dilution. AT activity remained significantly reduced from baseline levels over the postoperative period, recovering to baseline levels within 5 days, on average.
In this population of patients, MACE was associated with factors that have been previously associated with MI or mortality including recent MI, peripheral vascular disease, longer perfusion time, use of intraaortic counterpulsation, and red blood cell transfusion.7–10 The clinical model of MACE generated from this cohort had modest predictive value, similar to prior clinical models.9 The addition of AT activity on POD 2 and beyond to the model modestly improved model performance. However, the clinical value of AT activity as a predictor of MACE is limited by the majority of adverse events that comprised MACE occurring before POD 2. Specifically, the majority of MACE events were MI, which was typically manifested as the peak cTnI level occurring on POD 1. Rather, it may be that lower postoperative AT activity may be a consequence of more extensive surgery and other factors that are associated with increased incidence of MACE, although such assertion cannot be proven in this observational cohort. We cannot exclude the possibility that lower AT levels may have enhanced postoperative MACE.
Increased risk of adverse cardiovascular events has been associated with lower levels of AT in other critically ill populations, such as patients with severe sepsis.11–13 These observations have prompted clinical trials of supplemental AT administration.14–17 Although several trials have shown survival benefit, a meta-analysis of 20 trials encompassing 3458 patients failed to show a survival benefit of administration of AT to critically ill patients.18
There are limited data regarding AT activity and adverse outcomes after cardiac surgery. The majority of studies describe use of AT for “heparin resistance” during CPB and lack postoperative clinical outcome data.19–25 A single well-conducted observational study of 647 patients evaluated the association between preoperative and immediate postoperative AT levels and outcomes in cardiac surgery patients. Low levels of AT activity upon ICU arrival were associated with prolonged ICU stay, higher rate of reexploration for bleeding, thromboembolism, and adverse neurologic sequelae.5 Our study examined a longer time period that encompassed the period of MACE occurrence and the recovery of AT levels, thus providing additional insights. Importantly, we replicated the finding that AT level after CPB is associated with clinical factors indicative of longer, more extensive procedures, perhaps with more hemodilution.
Although our study contributes to our understanding of the role AT plays in the perioperative period, we are concerned by the absence of a temporal correlation between lower AT activity and MACE in our cohort, likely indicating that AT level is not an important determinant of thrombotic complications such as MI, stroke, and graft occlusion in the cardiac surgical setting. Thus, our observational cohort cannot directly address any putative biological mechanism for the role of AT in MACE generation.
In a large cohort of patients undergoing CABG surgery, postoperative AT activity was independently associated with MACE. Because this occurs at time points predominantly after the MACE event, the clinical utility of AT as a biomarker of risk remains unknown.
There are four potential conflicts of interest. Dr. Garvin was the recipient of a Research Fellowship funded by Talecris Biotherapeutics, which allowed time for generation of this and other articles. Dr. Chen is an employee of Talecris Biotherapeutics and performed the majority of the analyses. To prevent the appearance or substance of conflict, Simon Body personally directed the conduct of all analyses and confirmed the conduct of the analyses by reviewing the code and output of all analyses. In addition, Simon Body personally reran the important components of the analyses to confirm the findings and can attest that there was no potential for conflict in the analysis phase. Dr. Body received a total of <$5000 to allow his time for travel to Talecris in North Carolina to coordinate the analyses. Talecris also paid for the costs of antithrombin analyses performed by Charles River Laboratories in Ontario, Canada, but had no opportunity to intervene in these analyses. In brief, we believe there is no conflict of interest in the conduct of this study.
1. Maclean PS, Tait RC. Hereditary and acquired antithrombin deficiency: epidemiology, pathogenesis and treatment options. Drugs 2007;67:1429–40
2. Levy JH, Despotis GJ, Szlam F, Olson P, Meeker D, Weisinger A. Recombinant human transgenic antithrombin in cardiac surgery: a dose-finding study. Anesthesiology 2002;96:1095–102
3. Sanson BJ, Simioni P, Tormene D, Moia M, Friederich PW, Huisman MV, Prandoni P, Bura A, Rejto L, Wells P, Mannucci PM, Girolami A, Buller HR, Prins MH. The incidence of venous thromboembolism in asymptomatic carriers of a deficiency of antithrombin, protein C, or protein S: a prospective cohort study. Blood 1999;94:3702–6
4. Simioni P, Sanson BJ, Prandoni P, Tormene D, Friederich PW, Girolami B, Gavasso S, Huisman MV, Buller HR, Wouter ten Cate J, Girolami A, Prins MH. Incidence of venous thromboembolism in families with inherited thrombophilia. Thromb Haemost 1999;81:198–202
5. Ranucci M, Frigiola A, Menicanti L, Ditta A, Boncilli A, Brozzi S. Postoperative antithrombin levels and outcome in cardiac operations. Crit Care Med 2005;33:355–60
6. Ranucci M, Ditta A, Boncilli A, Cotza M, Carboni G, Brozzi S, Bonifazi C, Tiezzi A. Determinants of antithrombin consumption in cardiac operations requiring cardiopulmonary bypass. Perfusion 2004;19:47–52
7. Bradshaw PJ, Jamrozik K, Le M, Gilfillan I, Thompson PL. Mortality and recurrent cardiac events after coronary artery bypass graft: long term outcomes in a population study. Heart 2002;88:488–94
8. Charlesworth DC, Likosky DS, Marrin CA, Maloney CT, Quinton HB, Morton JR, Leavitt BJ, Clough RA, O'Connor GT. Development and validation of a prediction model for strokes after coronary artery bypass grafting. Ann Thorac Surg 2003;76:436–43
9. Koch CG, Weng YS, Zhou SX, Savino JS, Mathew JP, Hsu PH, Saidman LJ, Mangano DT. Prevalence of risk factors, and not gender per se, determines short- and long-term survival after coronary artery bypass surgery. J Cardiothorac Vasc Anesth 2003;17:585–93
10. van Brussel BL, Plokker HW, Voors AA, Ernst JM, Ernst NM, Knaepen PJ, Koomen EM, Tijssen JG, Vermeulen FE. Multivariate risk factor analysis of clinical outcome 15 years after venous coronary artery bypass graft surgery. Eur Heart J 1995; 16:1200–6
11. Martinez MA, Pena JM, Fernandez A, Jimenez M, Juarez S, Madero R, Vazquez JJ. Time course and prognostic significance of hemostatic changes in sepsis: relation to tumor necrosis factor-alpha. Crit Care Med 1999;27:1303–8
12. Sakr Y, Reinhart K, Hagel S, Kientopf M, Brunkhorst F. Antithrombin levels, morbidity, and mortality in a surgical intensive care unit. Anesth Analg 2007;105:715–23
13. Wilson RF, Mammen EF, Tyburski JG, Warsow KM, Kubinec SM. Antithrombin levels related to infections and outcome. J Trauma 1996;40:384–7
14. Baudo F, Caimi TM, de Cataldo F, Ravizza A, Arlati S, Casella G, Carugo D, Palareti G, Legnani C, Ridolfi L, Rossi R, D'Angelo A, Crippa L, Giudici D, Gallioli G, Wolfler A, Calori G. Antithrombin III (ATIII) replacement therapy in patients with sepsis and/or postsurgical complications: a controlled double-blind, randomized, multicenter study. Intensive Care Med 1998;24:336–42
15. du Cheyron D, Bouchet B, Bruel C, Daubin C, Ramakers M, Charbonneau P. Antithrombin supplementation for anticoagulation during continuous hemofiltration in critically ill patients with septic shock: a case-control study. Crit Care 2006;10:R45
16. Eid A, Wiedermann CJ, Kinasewitz GT. Early administration of high-dose antithrombin in severe sepsis: single center results from the KyberSept-trial. Anesth Analg 2008;107:1633–8
17. Kienast J, Juers M, Wiedermann CJ, Hoffmann JN, Ostermann H, Strauss R, Keinecke HO, Warren BL, Opal SM. Treatment effects of high-dose antithrombin without concomitant heparin in patients with severe sepsis with or without disseminated intravascular coagulation. J Thromb Haemost 2006;4:90–7
18. Afshari A, Wetterslev J, Brok J, Moller A. Antithrombin III in critically ill patients: systematic review with meta-analysis and trial sequential analysis. BMJ 2007;335:1248–51
19. Avidan MS, Levy JH, van Aken H, Feneck RO, Latimer RD, Ott E, Martin E, Birnbaum DE, Bonfiglio LJ, Kajdasz DK, Despotis GJ. Recombinant human antithrombin III restores heparin responsiveness and decreases activation of coagulation in heparin-resistant patients during cardiopulmonary bypass. J Thorac Cardiovasc Surg 2005;130:107–13
20. Conley JC, Plunkett PF. Antithrombin III in cardiac surgery: an outcome study. J Extra Corpor Technol 1998;30:178–83
21. Despotis GJ, Levine V, Joist JH, Joiner-Maier D, Spitznagel E. Antithrombin III during cardiac surgery: effect on response of activated clotting time to heparin and relationship to markers of hemostatic activation. Anesth Analg 1997;85:498–506
22. Kanbak M. The treatment of heparin resistance with antithrombin III in cardiac surgery. Can J Anaesth 1999;46:581–5
23. Lemmer JH Jr, Despotis GJ. Antithrombin III concentrate to treat heparin resistance in patients undergoing cardiac surgery. J Thorac Cardiovasc Surg 2002;123:213–7
24. Levy JH, Montes F, Szlam F, Hillyer CD. The in vitro effects of antithrombin III on the activated coagulation time in patients on heparin therapy. Anesth Analg 2000;90:1076–9
© 2010 International Anesthesia Research Society
25. Williams MR, D'Ambra AB, Beck JR, Spanier TB, Morales DL, Helman DN, Oz MC. A randomized trial of antithrombin concentrate for treatment of heparin resistance. Ann Thorac Surg 2000;70:873–7