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Aprotinin Prolongs Whole Blood Activated Partial Thromboplastin Time but Not Whole Blood Prothrombin Time in Patients Undergoing Cardiac Surgery

Despotis, George J. MD; Alsoufiev, Alexander MD; Goodnough, Lawrence T. MD; Lappas, Demetrios G. MD

Cardiovascular Anesthesia

Aprotinin is being used increasingly to limit cardiopulmonary bypass (CPB)-induced coagulation derangements. Since whole blood prothrombin time (PT) and activated partial thromboplastin time (APTT) assays are beneficial in the treatment of bleeding after CPB, we studied the potential effect of aprotinin on these whole blood assays. Blood specimens from 151 cardiac surgical patients were obtained in two phases: prior to heparin administration, before CPB, and subsequent to heparin neutralization after CPB. After collection, blood specimens were divided into two aliquots and mixed with either normal saline (NS) or aprotinin (A, 200 or 400 Kallikrein inhibiting units (KIU)/mL). Whole blood specimens were used to measure whole blood PT and APTT using CoaguChek Registered Trademark Plus instruments. Whole blood PT results were similar between normal saline (NS)- and aprotinin-spiked specimens before CPB (A, 12.9 +/- 1.5 s; NS, 12.8 +/- 1.5 s; P = 0.76) and after CPB (A, 17.5 +/- 2.4 s; NS, 17.7 +/- 2.4 s; P = 0.58). In contrast, whole blood APTT results were prolonged in aprotininspiked specimens prior to CPB (A, 63.3 +/- 32.2 s; NS, 38.6 +/- 16.3 s; P < 0.0001) and after CPB (A, 65.9 +/- 23.7 s; NS, 45.7 +/- 14.4 s; P < 0.0001). A dose-dependent prolongation of whole blood APTT by aprotinin was demonstrated by a greater mean difference in APTT (P = 0.0001) between specimens spiked with NS or 200 KIU (17.5 +/- 12.2 s) vs 400 KIU (27.8 +/- 21.5 s) of aprotinin. A greater mean difference in APTT values (P < 0.0001) between NS- and aprotinin-spiked specimens was demonstrated when comparing APTT measurements between specimens obtained from patients receiving (H) or not receiving (NH) heparin preoperatively (H, 36.5 +/- 18 s; NH, 18.1 +/- 21.9 s). Our data demonstrate that aprotinin-mediated prolongation of whole blood APTT is dose-dependent and enhanced by heparin. We conclude that when the whole blood APTT assay is used to assess heparin anticoagulant effect or coagulation factor levels after CPB, results should be interpreted with caution in the setting of concurrent aprotinin administration.

(Anesth Analg 1995;81:919-24)

Departments of Anesthesiology, Internal Medicine, Pathology, Washington University School of Medicine, St. Louis, Missouri.

Supported in part by a research grant from Boerhinger Mannheim Inc.

Accepted for publication June 13, 1995.

Address correspondence and reprint requests to George Despotis, MD, Division of Cardiothoracic Anesthesiology, Department of Anesthesiology, Box 8054, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.

After postcardiopulmonary bypass (CPB), a prompt assessment of coagulation allows targeted therapy for acquired hemostatic defects. In a recent study, the use of point-of-care assays for platelet count, whole blood prothrombin time (PT) and activated partial thromboplastin time (APTT), coupled with a transfusion and hemostasis algorithm, improved the management of post-CPB microvascular bleeding [1].

Use of aprotinin significantly reduces bleeding and blood product use in cardiac surgical patients [2,3]. Aprotinin has been shown to prolong celite but not kaolin activated clotting time (ACT) [4,5] and aprotinin has also been shown to affect APTT measurements using several reagents [6]. Other reports indicate that aprotinin may also affect the extrinsic pathway [7,8]. Whole blood PT and APTT assays using a thromboplastin reagent (whole blood PT) and a partial thromboplastin reagent with a bean phosphatide activator (whole blood APTT) assess the extrinsic and intrinsic pathways, respectively. Any potential effects of aprotinin on these assays are important, and therefore we studied the effect of aprotinin on whole blood PT and APTT assays.

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Methods

Excess or waste blood from specimens obtained for routine coagulation analysis from 151 consecutive adult patients undergoing cardiac surgery requiring CPB was used in this study. Under the guidelines of our Institutional Human Studies Committee, informed consent was not required for preexisting specimens. All patients were anesthetized with an opioid-based technique and the anesthetic was supplemented with inhaled anesthetics, muscle relaxants, and benzodiazepines. Extracorporeal circulation was accomplished with a Biomedicus Registered Trademark centripetal pump (Medtronic Cardiopulmonary, Anaheim, CA), a Bentley Registered Trademark membrane oxygenator (Baxter Bentley, Irvine, CA), and systemic hypothermia was maintained at 28 degrees C during cardioplegia. The CPB system was routinely primed with 1.5 L of Plasmalyte Registered Trademark, 50 mEq of sodium bicarbonate, 25 g of Mannitol, and 5000 U of porcine heparin. Systemic anticoagulation for CPB was accomplished with porcine heparin and was administered based on on-site measurements of heparin dose response (HDR Registered Trademark), heparin concentration, and kaolin ACT using the Hepcon Registered Trademark instrument (Medtronic Hemotec, Englewood, CO), as previously described [9]. After rewarming the patient to 37 degrees C, extracorporeal circulation was discontinued and heparin was neutralized with protamine. The protamine dose was determined based on the whole blood heparin concentration measured prior to discontinuation of CPB (1.3 mg of protamine per mg of residual heparin).

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Study Design

To assess the effect of aprotinin on whole blood PT and APTT measurements before and after CPB, the study was divided into two phases. Phase I was designed to evaluate the effect of aprotinin on these assays before CPB and was performed with blood specimens obtained from patients prior to heparin administration for CPB. Blood specimens were divided into two aliquots and inserted into vials containing either normal saline (NS) or one of two known amounts of aprotinin (Miles Inc., West Haven, CT) yielding final concentrations of either 28 or 56 micro gram/mL. These concentrations are equivalent to 200 or 400 kallikrein inhibiting units (KIU)/mL, respectively, which are typically seen with administration of the full Hammersmith regimen [2]. Phase II was designed to evaluate the effect of aprotinin on whole blood PT and APTT measurements after CPB and was performed with blood specimens obtained from patients after administration of protamine subsequent to discontinuation of CPB. As with Phase 1, blood specimens were divided into two aliquots and inserted into vials containing either NS or aprotinin (A) yielding either 28 (200 KIU) or 56 (400 KIU) micro gram/mL final concentrations. After specimens were mixed, whole blood PT and APTT were then measured in duplicate.

In addition to comparing the effect of aprotinin on whole blood PT and APTT measurements between the pre-CPB and post-CPB intervals, results from three subsets of patients were used for additional analyses. To examine the potential impact of in vivo heparin on the effect of aprotinin on whole blood PT and APTT measurements, prolongations of whole blood PT and APTT by aprotinin were compared between specimens obtained from patients receiving (H, n = 52) and not receiving (NH, n = 99) heparin infusions preoperatively. To determine whether the effect of aprotinin is dose-dependent, prolongations in whole blood PT and APTT measurements were compared between specimens, which were spiked with either 28 or 56 micro gram/mL of aprotinin (28 micro gram/mL; n = 75; 56 micro gram/mL, n = 76), obtained from two subsets of patients. To compare the effect of in vitro versus ex vivo aprotinin on whole blood PT and APTT, a small subset of patients (n = 7) who received aprotinin infusion as part of their management were included in our analysis. In these patients, whole blood PT and APTT were assayed prior to administration of aprotinin (NS-spiked), after in vitro addition of aprotinin (200 KIU/mL) and after systemic administration of aprotinin (bolus and infusion).

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Coagulation Assays

Single blood specimens obtained from either radial and/or femoral intraarterial catheters after removal of six dead space volumes were used for coagulation analysis by on-site, whole blood assays. Whole blood PT and APTT were measured using four CoaguChek Registered Trademark Plus instruments (Boehringer Mannheim Diagnostics, Indianapolis, IN) that use disposable plastic reagent cartridges as described previously by Lucas et al. [10].

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Statistical Analysis

Duplicate measurements of whole blood PT and APTT were expressed as mean values +/- SD. Student's paired t-test was used to compare whole blood PT and APTT between the pre-CPB and post-CPB periods in aprotinin-treated and control specimens within each patient, and to compare the effect of in vitro aprotinin versus ex vivo aprotinin on whole blood PT and APTT values in a small series of patients (n = 7). To simplify our comparisons, values representing the prolongation of whole blood PT and APTT measurements by aprotinin were calculated using the following formula: aprotinin-mediated prolongation = aprotinin-spiked measurements - control measurements (NS-spiked). Within each intraoperative interval (pre-CPB and post-CPB), Student's unpaired t-test was used to compare the effect of aprotinin concentration (200 or 400 KIU/mL) on mean whole blood PT and APTT measurements using specimens from two subsets of patients. To evaluate the effect of heparin on aprotinin-mediated prolongation of whole blood PT and APTT, mean PT and APTT measurements obtained prior to CPB were compared between aprotinin-treated specimens obtained from patients receiving or not receiving heparin preoperatively using Student's unpaired t-test. In addition, mean differences between control specimens and aprotinin-spiked specimens were compared between the pre-CPB and post-CPB periods in patients who received preoperative heparin infusions using paired t-test. Wilcoxon signed rank (paired data) and ranked sum tests (unpaired data) or t-test for unequal variances (paired or unpaired data) were used to compare mean whole blood PT and APTT when unequal variances were demonstrated.

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Results

Effect of Aprotinin on Whole Blood PT

Whole blood PT results were similar between NS- and aprotinin-spiked specimens before CPB (A, 12.9 +/- 1.5 s; NS, 12.8 +/- 1.5 s; P = 0.76). Similarly, whole blood PT results were not influenced by aprotinin after CPB (A, 17.5 +/- 2.4 s; NS, 17.7 +/- 2.4 s; P = 0.58). Preoperative use of heparin did not influence whole blood PT results which were similar between NS- and aprotinin-spiked specimens before CPB and after CPB Table 1.

Table 1

Table 1

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Effect of Aprotinin on Whole Blood APTT: Impact of Aprotinin Dose with Each Collection Period

Whole blood APTT results were prolonged in aprotinin-spiked specimens prior to CPB (A, 63.3 +/- 32.2 s; NS, 38.6 +/- 16.3 s; P < 0.0001) and after CPB (A, 65.9 +/- 23.7 s; NS, 45.7 +/- 14.4 s; P < 0.0001) when compared to NS-spiked specimens. A dose-dependent prolongation of whole blood APTT by aprotinin was demonstrated by a greater mean difference in APTT (P = 0.0001) between specimens spiked with NS or 200 KIU/mL (17.5 +/- 12.2 s) vs 400 KIU/mL (27.8 +/- 21.5 s) of aprotinin. In specimens obtained prior to CPB, aprotinin prolonged whole blood APTT in a dose-dependent fashion, as demonstrated by a greater mean difference in APTT values (P = 0.007) between NS-spiked specimens and specimens spiked with 400 KIU/mL (29.3 +/- 26.9 s) when compared to 200 KIU/mL (19.5 +/- 15.0 s) of aprotinin. Aprotinin also prolonged whole blood APTT in a dose-dependent fashion in the post-CPB period as demonstrated by a greater mean difference in APTT values (P = 0.007) between NS-spiked specimens and specimens spiked with 400 KIU/mL (26.3 +/- 13.5 s) vs 200 KIU/mL (15.2 +/- 7.7 s) of aprotinin.

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Effect of Aprotinin on Whole Blood APTT in Patients Receiving or Not Receiving Heparin Infusion Preoperatively

In patients not receiving heparin preoperatively (n = 99), aprotinin-mediated prolongation of mean whole blood APTT tended to be greater after CPB when compared to before CPB (pre-CPB, 18.1 +/- 21.9 s; post-CPB, 22.2 +/- 13.3 s; P = 0.1). As illustrated in Figure 1, prolongation in whole blood APTT by aprotinin was also dose-dependent, as demonstrated by a greater mean difference in APTT values between NS-spiked specimens and specimens spiked with 400 KIU/mL of aprotinin prior to CPB (400 KIU, 24.1 +/- 30.1 s; 200 KIU, 12.3 +/- 3.7 s; P = 0.009) and after CPB (400 KIU, 29.1 +/- 14.4 s; 200 KIU, 15.7 +/- 7.9 s; P < 0.0001).

Figure 1

Figure 1

In patients receiving heparin preoperatively (n = 52), prolongation of whole blood APTT by aprotinin was more pronounced prior to CPB when compared to after CPB, as demonstrated by a greater mean difference between APTT measurements (pre-CPB, 36.5 +/- 17.9 s; post-CPB, 18.4 +/- 10.1 s; P < 0.0001) in specimens containing heparin. Although prolongation of whole blood APTT by aprotinin was dose-dependent in the post-CPB period, as demonstrated by a greater mean difference in APTT values between NS-spiked specimens and specimens spiked with 400 KIU/mL of aprotinin (400 KIU, 21.8 +/- 10.7 s; 200 KIU, 14.3 +/- 7.5 s; P < 0.007), Figure 2 illustrates that this was not evident in specimens obtained prior to CPB from patients receiving preoperative heparin infusion (400 KIU, 37.8 +/- 18.0 s; 200 KIU, 34.9 +/- 18.2 s; P < 0.56).

Figure 2

Figure 2

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Effect of Heparin on Aprotinin-Mediated Prolongation of Whole Blood APTT

During the pre-CPB period, a greater mean difference in APTT values was demonstrated (P < 0.0001) between NS- and aprotinin-spiked specimens when comparing specimens from patients receiving heparin preoperatively to specimens obtained from patients not receiving heparin preoperatively (H, 36.5 +/- 18 s; NH, 18.1 +/- 21.9 s). During the post-CPB period, the mean difference in APTT values between NS- and aprotinin-spiked specimens was similar (P = 0.08) when comparing specimens from patients receiving heparin (H) preoperatively to specimens obtained from patients not receiving heparin preoperatively (H, 18.4 +/- 10 s; NH, 22.2 +/- 13.3 s).

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Effect of In Vivo Versus Ex Vivo Aprotinin on Whole Blood PT and APTT

Whole blood PT measurements were similar between NS-spiked specimens (14.1 +/- 3.3 s) and specimens with either in vitro (14.2 +/- 3.5 s; p = 0.5) or ex vivo (15.5 +/- 4.4 s; P = 0.3) aprotinin. In contrast, whole blood APTT was prolonged by both in vitro (51.5 +/- 7.2 s; p = 0.01) and ex vivo (51 +/- 19.5 s; P = 0.03) aprotinin when compared to whole blood APTT values in NS-spiked specimens (34.1 +/- 5.9 s). Aprotinin added in vitro (200 KIU) prolonged whole blood APTT to the same degree as ex vivo aprotinin as demonstrated by a similar mean difference in APTT values between aprotinin-spiked specimens and NS-spiked specimens (in vitro, 17.4 +/- 3.9 s; ex vivo, 16.8 +/- 21.7 s; P = 0.94).

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Discussion

Transfusion of blood and blood products is currently scrutinized due to increased awareness of the risks and costs associated with transfusion. Consequently, strategies such as the use of point-of-care assays (whole blood PT, APTT, and platelet count) to improve diagnosis and treatment of microvascular bleeding have been studied [1]. Additionally, antifibrinolytic agents such as epsilon-aminocaproic acid [11,12], tranexamic acid [13,14], and aprotinin [2,3] are commonly used to reduce bleeding and transfusion requirements. The mechanism in common for these three agents is inhibition of the plasminogen/plasmin pathway. In addition to plasmin inhibition [15], aprotinin also has significant antiinflammatory properties (e.g., kallikrein and complement inhibition) [15], may protect platelet vWF activity [16] as well as platelet glycoprotein lb [17] and IIb/IIIa receptors [2] from proteolytic degradation and is a competitive inhibitor of the factor VIIa-tissue factor complex [7].

The ACT is routinely used to assess adequacy of heparin anticoagulation before and during extracorporeal circulation. Previous in vitro [4] and ex vivo [5] data demonstrate prolongation of celite, but not kaolin ACT, by aprotinin. The PT evaluates the extrinsic and common pathways of the coagulation system; significant prolongation occurs when there is a deficiency of factors VII, V, X, prothrombin, or fibrinogen, or when there is a circulating inhibitor. Our data illustrate that the whole blood PT assay is not affected by concentrations of aprotinin that approximate those commonly observed with the full Hammersmith regimen [18]. This is in contrast to previous reports indicating that aprotinin may affect extrinsic coagulation [7,8]. The APTT evaluates the intrinsic and common pathways; significant prolongation occurs when there is deficiency of factors XII, XI, IX, VIII, X, V, prothrombin, and fibrinogen or when there is a circulating inhibitor. Our data demonstrate that aprotinin significantly prolongs the whole blood APTT assay. Although our analysis did not encompass a dose-range within each patient, a dose-dependent effect was seen with incrementally larger doses (i.e., from 200 to 400 KIU) of aprotinin. Greater prolongations in whole blood APTT by aprotinin were evident in specimens obtained prior to CPB in patients receiving heparin infusions preoperatively. Although reports have suggested that aprotinin may have anticoagulant properties [19,20], other reports reveal that prolonged celite ACT values may reflect aprotinin's ability to inhibit activators of the ACT assay [21,22]. Although prolongations in whole blood APTT by aprotinin may reflect aprotinin's anticoagulant properties, a more feasible explanation may be that aprotinin inhibits the bean phosphatide activator used in the whole blood APTT assay. This is consistent with a previous study which demonstrated that aprotinin prolongs APTT measurements using several reagents and that kaolin-activated APTT reagents may be less affected by aprotinin [6].

An extensive comparison of APTT measurements between the whole blood assay and standard reference methods has been performed previously in a four-center investigation [23]. The response of whole blood APTT values to heparin has also been evaluated recently [24]. This previous study revealed that both whole blood and laboratory APTT measurements respond similarly to incrementally larger doses of in vitro heparin, and demonstrated a good correlation between whole blood APTT values and anti-Xa heparin concentration [24]. Since aprotinin affects the whole blood APTT assay, the response of whole blood APTT to heparin is difficult to interpret in the presence of aprotinin. In addition, whole blood APTT results should be interpreted with caution when trying to identify coagulation factor deficiencies [25] in the setting of concurrent aprotinin administration. As previously described, whole blood PT and APTT assays are an important accompaniment to the use of a transfusion algorithm in the treatment of excessive bleeding after CPB. Since aprotinin does not affect whole blood PT measurements, this assay can be used in this setting. Our data demonstrate that aprotinin has a variable effect on whole blood APTT in the post-CPB setting. In addition, aprotinin concentrations can vary significantly using the full Hammersmith dosing regimen [2]. Therefore, it would be difficult to quantitatively adjust whole blood APTT values in an attempt to factor in the impact of aprotinin on these measurements. On this basis, whole blood APTT results should not be incorporated into algorithms that direct hemostatic transfusion in the setting of concurrent aprotinin administration.

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