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Anesth Analg 2000;90:1076-1079
© 2000 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

The In Vitro Effects of Antithrombin III on the Activated Coagulation Time in Patients on Heparin Therapy

Jerrold H. Levy, MD*, Felix Montes, MD*, Fania Szlam, MMSc*, and Christopher D. Hillyer, MD{dagger}

Departments of *Anesthesiology and {dagger}Pathology, Emory University School of Medicine, Division of Cardiothoracic Anesthesiology and Critical Care, Emory Healthcare, Atlanta, Georgia

Address correspondence and reprint requests to Jerrold H. Levy, MD, Department of Anesthesiology, Emory University Hospital, 1364 Clifton Rd., N.E., Atlanta, GA 30322. Address e-mail to jerrold_levy{at}emory.org


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Heparin requires antithrombin III (AT) to achieve anticoagulation, and patients on continuous small-dose heparin preoperatively experience decreased levels of AT-causing heparin resistance. When this occurs, 2–4 units of fresh frozen plasma (~1000 units of AT) are often administered to increase AT levels and restore heparin responsiveness. We evaluated purified human AT concentrate (Thrombate III; Bayer, Inc., Elkhart, IN) to restore in vitro anticoagulation responses in patients receiving heparin. Blood samples were obtained from cardiac surgery patients including 22 patients receiving heparin and 21 patients not receiving heparin preoperatively. Heparin was added to blood in final concentrations of 4.1, 5.4, and 6.8 U/mL (equivalent to 300, 400, and 500 U/kg), and kaolin-activated clotting times (ACTs) were determined with and without AT at a final concentration of 0.2 units/mL to mimic fresh frozen plasma administration. The mean duration of preoperative heparin therapy was 4.0 days (range 2–10 days). AT activity was 69% ± 9% in patients receiving heparin and 92% ± 8% in patients not receiving heparin (P < 0.01). Heparin >4.1 U/mL failed to further increase ACT values in all patients. Attempts to increase ACT in patients receiving heparin may require supplemental AT administration. Purified AT even in small doses significantly prolongs the ACT response to heparin.

Implications: In vitro addition of antithrombin III (0.2 U/mL) to heparinized blood samples (4.1–6.8 units of heparin/mL) from patients on previous heparin therapy increases sensitivity to supplemental heparin as reflected by significantly prolonged activated clotting time.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Cardiopulmonary bypass (CPB) results in the interface of blood with a large foreign surface that activates the hemostatic cascade. Heparin is the current drug of choice for systemic anticoagulation because of its prompt onset of action, rapid reversal with protamine sulfate, and low cost. Heparin inhibits clotting by binding to antithrombin III (AT), and acts as a catalyst to augment AT inhibition of target serum proteinases, especially thrombin and Xa. In patients receiving heparin, a relative resistance to heparin as manifested by decreased responsiveness of the activated clotting time (ACT) has been reported. Heparin resistance is defined as the inability of >=500 U/kg of IV heparin to produce an ACT value of at least 480 s (1).

A decreased response to increasing doses of heparin is believed to be secondary to a decrease in AT level. The time course for developing AT deficiency is unknown, but it likely develops within the first 12 h of heparin therapy. Infusion of 2–4 units of fresh frozen plasma (FFP) has been recommended as an indirect method of increasing AT levels when patients are refractory to additional heparin boluses (2). The natural variability of AT content, the time required to thaw FFP, the expense, and inherent risks of transmissible diseases make other alternatives more desirable. Purified AT concentrates are available, but data that establish the dose of AT concentrate necessary to restore responsiveness are lacking, and 1000 units is currently used as an initial dose for replacement (3). The purpose of this investigation was to assess the ability of an in vitro AT supplement of 0.2 U/mL (corresponding to 1000 units AT or ~2–4 units of FFP in vivo) to restore heparin responsiveness (as measured by ACT) in blood obtained from cardiac surgery patients on continuous preoperative IV heparin (1000 U/h).


   Methods
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 Abstract
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 Methods
Results
 Discussion
 References
 
After institutional approval and informed consent, blood samples from 43 adult patients scheduled for elective cardiac surgery requiring CPB were evaluated. Both the duration of IV heparin therapy and concomitant drug administration were recorded. Baseline platelet count, prothrombin time, and partial thromboplastin time were recorded. Patients were divided into two groups: heparin-treated patients (n = 22) on preoperative IV heparin infusions for at least 48 h (Group 1), and control patients (n = 21) who had not received preoperative IV heparin (Group 2). After the induction of anesthesia, but before the administration of IV heparin for aortic cannulation, blood samples were obtained from the arterial catheter.

Blood for measurement of AT activity was collected into plastic tubes containing sodium citrate 3.2% (9:1, vol/vol). Samples were immediately centrifuged at 2000g for 15 min at 21°C. Plasma supernate was retrieved into plastic tubes and stored at -80°C until testing was performed. AT levels were analyzed by using Coatest AntithrombinTM (Chromogenic AB, Mondal, Sweden) according to the manufacturer’s specifications.

Baseline kaolin ACT was measured intraoperatively on blood specimens drawn from heparin-treated and control patients. Aliquots of blood (0.4 mL) from both groups were mixed with heparin (1.65–2.8 µL; 1000 U/mL) to achieve a final concentration of 4.1, 5.4, and 6.8 U/mL of bovine lung heparin (Upjohn, Kalamazoo, MI) (equivalent to 300, 400, and 500 U/kg body weight, respectively). AT (Thrombate IIITM; Bayer, Inc., Elkhart, IN) at a final concentration of 0.2 U/mL (1.5 µL) was added to heparinized blood samples as described. The addition of heparin and AT resulted in less than 1% dilution, compared with baseline samples. Control samples (except baseline) had only heparin added (no AT), which resulted in less than 0.25% dilution. ACTs were measured in duplicate, immediately after the blood was drawn by using TMMedtronic-Hemotec high-range cartridges with kaolin and run on Hemotec ACT machines (Hemotec-Medtronic, Englewood, CO). All ACTs were kaolin-based because of the small volumes of blood needed for the testing and because of the quick response time of the Medtronics ACT system.

All data were expressed as mean ± SD of the mean. Serial data for each group were evaluated by the repeated-measures analysis of variance, followed by the paired t-test with the Bonferroni correction. Differences between groups were compared by using one-way analysis of variance. A P value equal to or less than 0.05 was considered significant. Regression analysis using the method of least squares was used to evaluate the relationship between AT concentration and in vitro postheparin ACT in blood specimens with and without addition of AT.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Demographic profiles for subjects in the heparin-treated and control patients were equivalent (Table 1). All patients in Group 1 were treated with preoperative IV heparin, 1000 U/h, for unstable angina for an average of 4.04 days (range 2–10 days). The heparin infusion was continued until surgery. Eleven patients in the heparin-treated group and 8 patients in the control group received aspirin until the day before surgery.


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  		Table 1. Patient Demographic Data
 
Baseline ACT in patients receiving preoperative IV heparin was 160 ± 37 s. Anticoagulation of these blood specimens to a final heparin concentration of 4.1, 5.4, and 6.8 U/mL resulted in the increase of ACT values to 453 ± 130, 478 ± 108, and 496 ± 106 s, respectively (Fig. 1). The addition of AT to the anticoagulated blood samples resulted in a significant increase in the ACT to 567 ± 110, 612 ± 138, and 678 ± 146 s (P < 0.01) (Fig. 1). Patients not receiving preoperative IV heparin had a mean baseline ACT of 130 ± 22 s, and heparin added at concentrations of 4.1, 5.4, and 6.8 U/mL resulted in an increase in ACT to 568 ± 134, 607 ± 142, and 628 ± 152 s, respectively (Fig. 2). AT added to the anticoagulated blood specimens in the control groups also resulted in a significant increase in ACT to 652 ± 153, 711 ± 162, and 761 ± 155 s (P < 0.05) (Fig. 2).



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  		Figure 1. In vitro activating clotting time (ACT) responses in seconds (sec) with and without 0.2 U/mL of antithrombin III (AT) added to blood in patients receiving heparin preoperatively. The addition of heparin to the baseline samples produced significant prolongations of the ACT in all of the samples studied (P < 0.01 versus baseline); however, there were no significant differences in ACTs among incremental heparin doses after 4.1 U/mL. The addition of AT to each heparinized sample significantly increased the ACT for two highest concentrations of heparin (5.4 and 6.8 U/mL) (** P < 0.05 AT versus no AT).

 


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  		Figure 2. In vitro activating clotting time (ACT) responses in seconds (sec) with and without 0.2 U/mL of antithrombin III (AT) added to blood in patients not receiving heparin preoperatively. The addition of heparin to the baseline samples produced significant prolongations of the ACT in all of the samples studied (P < 0.01 versus baseline); however, there were no significant differences in ACTs among incremental heparin doses after 4.1 U/mL. The addition of AT to each heparinized sample significantly increased the ACT for each concentrations of heparin (** P < 0.01 AT versus No AT).

 
Figures 1 and 2 demonstrate that anticoagulation to target heparin concentrations resulted in a significant increase in ACT, compared with baseline in both groups (P < 0.01); however, there were no significant differences in ACTs among incremental heparin doses after 4.1 U/mL. The heparin dose/ACT response was not linear. When AT was added to each target concentration of heparin, a significantly larger increase in the ACT was observed in the heparin-treated patients than in the controls (Figs. 1 and 2). AT activity was decreased in patients receiving preoperative IV heparin therapy and was normal in those patients not receiving IV heparin (Table 1). The difference between AT activity levels in both groups was statistically significant (P < 0.01). The relationship between preoperative plasma AT activity and prolonged ACT after in vitro heparinization was examined in both groups. No relationship was identified between preoperative AT activity and ACT measured after heparinization with or without the addition of AT.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We found reduced ACT responses in vitro to heparin in patients receiving IV heparin therapy that could be increased after AT supplementation. Although patients receiving heparin had lower AT levels compared with controls, both groups of patients demonstrated increased ACTs once AT was added. The in vitro dose of 0.2 units of AT per mL of blood corresponds to approximately 1000 units AT concentrate transfusion for a 70-kg adult. We chose that dose because it is approximately equivalent to the AT concentration of 4 units (~225 mL/U, 1 U/mL) of FFP (at a cost of approximately US $200 based on our blood bank charges) and has been reported as a therapeutic approach during CPB and coronary artery bypass grafting (2,4). The heparin concentrations studied corresponded to conventional doses of heparin (300–500 U/kg) administered for CPB.

We also found that concentrations of heparin greater than 4.1 U/mL (approximately equivalent to a dose of 300 U/kg) failed to produce statistically significant augmentation of ACTs (Figs. 1 and 2). This is different from the linear heparin-ACT response previously described by Bull et al. (5,6). The lack of linearity of our dose response curve is likely related to the intrinsic AT activity level required for the anticoagulant effect of heparin administered. The above heparin doses were selected because they represent anticoagulation protocols practiced among institutions and in vivo heparin concentrations that would be achieved if standard heparin dosing protocols are used.

We performed this study based on previous reports that patients receiving IV infusions of heparin before cardiac surgery develop varying degrees of heparin resistance. Staples et al. (1) observed the relative risk of heparin insensitivity or tachyphylaxis to be three times higher in patients receiving preoperative IV heparin. Heparin resistance is manifested as an attenuated increase in ACT after standard heparin dose (300–500 U/kg) to facilitate CPB. The reasons for heparin resistance are incompletely understood with many causes reported, including both preoperative heparin and nitroglycerin therapy that are frequently administered in patients with unstable angina (7–10). The results of the reported studies are conflicting and inconclusive, pointing to the need for additional investigations to elucidate the influence of drug therapies on the development of heparin resistance.

Results of our in vitro study also support previous reports demonstrating relative "heparin resistance" when patients on IV heparin present to cardiac surgery. The acute hemodilution during CPB is known to cause a further decrease in AT levels to approximately 50% of baseline values (11,12). Consequently, these patients may be at an even greater risk for inadequate anticoagulation during CPB. Thrombin generation and thrombin activity can occur despite adequate ACTs (>480 s) during CPB. A resultant low-grade activation of the clotting cascade potentially produced in part by the low levels of AT may cause consumption of coagulation proteins and platelets, leading to microvascular bleeding and ultimately resulting in increased requirements for transfusion of allogeneic blood products.

There are little data describing the use of AT as a therapeutic intervention in patients undergoing cardiac surgery. Hashimoto et al. (11) observed an increase in fibrinopeptide A during CPB despite standard heparinization and ACT monitoring. They also observed that AT given pre-CPB to pediatric patients not selected for heparin resistance prevented CPB associated decreases in AT, and thus prevented an elevation of fibrinopeptide A. Cardiac surgery patients, especially those receiving preoperative heparin, represent an important group for potential AT supplementation. The optimal dose of AT to improve heparin responses is not known; however, based on our in vitro study, 1000 units may sufficiently raise the ACT to an acceptable level. However, to normalize AT during coronary artery bypass grafting a dose of 75 U/kg may be required. Kanbak (3) described three heparin-resistant patients (ACT < 400 seconds) undergoing cardiac surgery who were successfully treated with 1000 units AT.

AT is a serine proteinase inhibitor that has inhibitory effects on multiple coagulation proteinases (thrombin, factors Xa, IXa, XIa, XIIa, plasmin, and kallikrein) and plays a key regulatory role in the natural control of thrombosis in vivo. The interaction of heparin with AT accelerates the rate of coagulant inactivation by reducing the half-life of coagulant proteinases. The efficiency of target proteinase inactivation is related to AT concentration, AT activity, and the molecular weight of heparin (13).

Despotis et al. (14) reported the effects of AT on ACT responses to heparin from normal volunteers, after the in vitro addition of AT, and after dilution with AT-deficient plasma. Despotis et al. (14) also collected plasma samples during CPB to measure the response of the kaolin ACT to heparin AT concentration and a battery of coagulation assays in 31 patients undergoing cardiac surgery. They reported linear relationships between kaolin and celite ACT slopes and AT concentrations (14) and found that the responsiveness of ACT to heparin is progressively reduced when the AT concentration decreases below 0.8 U/mL (80 U/dL) (14), which is the lower limit of normal plasma concentration.

The increasing use of IV heparin in patients with unstable angina is associated with reduced AT activity and heparin insensitivity. As shown by our in vitro study, supplemental doses of AT increase heparin dose responses, as measured by ACT values, and offer a novel therapeutic maneuver to improve anticoagulation responses. Despite the widespread practice of administering supplemental heparin to increase the ACT, this was not effective in our investigation. Additional studies are needed to further evaluate the therapeutic potential of AT concentrate for patients requiring anticoagulation for CPB surgery, as well as nonsurgery patients requiring prolonged infusion of IV heparin, because AT is one of many factors affecting ACT responses in vivo.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Staples MH, Dunton TF, Karlson KJ, et al. Heparin resistance after preoperative heparin therapy or intraaortic balloon pumping. Ann Thorac Surg 1994;57:1211–6.[Abstract]
  2. Sabbagh AH, Chung GK, Shuttleworth P, et al. Fresh frozen plasma: a solution to heparin resistance during cardiopulmonary bypass. Ann Thorac Surg 1984;37:466–8.[Abstract]
  3. Kanbak M. The treatment of heparin resistance with antithrombin III in cardiac surgery. Can J Anesth 1999;46:581–5.[Web of Science][Medline]
  4. Barnette RE, Shupak RC, Pontius J, Rao AK. In vitro effect of fresh frozen plasma on the activated coagulation time in patients undergoing cardiopulmonary bypass. Anesth Analg 1988;67:57–60.[Abstract/Free Full Text]
  5. Bull MH, Huse WM, Bull BS. Evaluation of tests used to monitor heparin therapy during extracorporeal circulation. Anesthesiology 1975;43:346–53.[Web of Science][Medline]
  6. Bull BS, Huse WM, Brauer FS, Korpman RA. Heparin therapy during extracorporeal circulation. II. The use of a dose-response curve to individualize heparin and protamine dosage. Thorac Cardiovasc Surg 1975;69:685–9.
  7. Habbab MA, Haft JI. Heparin resistance induced by intravenous nitroglycerine. A word of caution when both drugs are used concomitantly. Arch Intern Med 1987;147:857–60.[Abstract/Free Full Text]
  8. Lepor NE, Amin DK, Berberian L, Shah PK. Does nitroglycerine induce heparin resistance? Clin Cardiol 1989;12:432–4.[Web of Science][Medline]
  9. Schoenenberger RA, Menat L, Weiss P, et al. Absence of nitroglycerine-induced heparin resistance in healthy volunteers. Eur Heart J 1992;13:411–4.[Abstract/Free Full Text]
  10. Anderson EF. Heparin resistance prior to cardiopulmonary bypass. Anesthesiology 1986;64:504–7.[Web of Science][Medline]
  11. Hashimoto K, Yamagishi M, Sasaki T, et al. Heparin and antithrombin III levels during pulmonary bypass: correlation with subclinical plasma coagulation. Ann Thorac Surg 1994;58:799–805.[Abstract]
  12. Zaidan JR, Johnson S, Brynes R, et al. Rate of protamine administration: its effect on heparin reversal and antithrombin recovery after coronary artery surgery. Anesth Analg 1986;65:377–80.[Abstract/Free Full Text]
  13. Rosenberg RD, Bauer KA, Marcum JA. Antithrombin III : the heparin-antithrombin system. In: Murano G, ed. Reviews of hematology: protease inhibitors of human plasma biochemistry and pathophysiology target. Westbury:PJD Publications Ltd., 1986:351–416.
  14. Despotis GJ, Levine V, Joist JH, et al. 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.[Abstract]
Accepted for publication January 12, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press