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Ann Thorac Surg 1999;68:473-478
© 1999 The Society of Thoracic Surgeons

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Original Articles

Aprotinin inhibits thrombin formation and monocyte tissue factor in simulated cardiopulmonary bypass

^a Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, USA
^b Department of Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA
^c Department of Chemical Engineering, University of Delaware, Newark, Delaware, USA

Address reprint requests to Dr Colman, Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad St, Philadelphia, PA 19140
e-mail: colmanr{at}astro.temple.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Aprotinin reduces perioperative bleeding after open heart surgery, primarily by inhibiting fibrinolysis. In addition, the drug has both procoagulant and anticoagulant effects that involve complex reactions of coagulation proteins and cells that are incompletely understood. This study tests the hypothesis that aprotinin has an anticoagulant effect on the extrinsic coagulation pathway.

Methods. Human heparinized blood was recirculated through a membrane oxygenator with and without high concentrations of aprotinin (18.4 µM). Serial plasma samples were obtained at intervals up to 240 minutes.

Results. Aprotinin significantly reduced the progressive increase in prothrombin fragments (F1.2) and thrombin-antithrombin complex beginning immediately. Aprotinin also significantly reduced monocyte expression of tissue factor and Mac-1. Aprotinin did not significantly reduce factor VII or factor VIIa.

Conclusions. During simulated cardiopulmonary bypass, aprotinin immediately inhibits kallikrein and thrombin formation via the intrinsic coagulation pathway. Later, aprotinin inhibits monocyte expression of tissue factor and the extrinsic coagulation pathway. The ability of aprotinin to inhibit monocyte tissue factor provides a means to reduce thrombin formation in blood aspirated from the wound during open heart surgery.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Aprotinin reduces postoperative bleeding and transfusion requirements and hastens restoration of bleeding times to the normal range after cardiopulmonary bypass (CPB) [1, 2]. However, a report associates aprotinin with higher rates of coronary arterial bypass graft occlusions, perioperative myocardial infarction, and perioperative mortality [3], although these changes were not statistically significant. The optimal dose and dosing scheme for the drug during open heart surgery are still under investigation [2, 4], and the use of topical aprotinin in the wound has been recommended [5]. Lastly, the mechanisms by which aprotinin affects the coagulation cascade are not completely understood [6, 7].

Aprotinin is a bovine serine protease inhibitor that has both procoagulant and anticoagulant properties. The drug inhibits plasmin directly rather than inhibiting the formation of plasmin (Ki = 0.07 nM) [6]. At the highest clinically used doses (8.5 µM), aprotinin also inhibits kallikrein (Ki = 36 nM) [8], but has no direct inhibitory action on thrombin (Ki = 61,000 nM). The principal prothrombotic effect of aprotinin is inhibition of fibrinolysis, which is accelerated both in the wound and perfusion circuit during open cardiac surgery [9, 10]. The drug also appears to attenuate platelet secretion and loss of responsiveness to adenosine diphosphate (ADP) [8], but does not inhibit platelet adhesion in vitro or in vivo [8, 11, 12]. Lastly, at very high concentrations (IC₅₀ = 55 µM), aprotinin partially inhibits purified factor VIIa-tissue factor complex in vitro [7], but its effect on plasma factor VIIa is unknown.

This study was designed to investigate the interaction of aprotinin with enzymes in the coagulation pathway. We were particularly interested in the possible anticoagulant activity of the drug in the surgical wound. We tested the hypothesis that, at high concentrations, aprotinin decreases thrombin formation by inhibiting constituents of the extrinsic coagulation pathway. This effect would be in addition to its known anticoagulant effect on the intrinsic pathway due to direct kallikrein inhibition.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
In vitro study of factor VIIa inhibition by aprotinin
Since a direct inhibitory effect of aprotinin on purified factor VIIa-factor complex had been reported [7], a preliminary in vitro study using pooled normal citrated plasma was performed. Aprotinin at final concentrations varying from 4.5 to 144 µM was added to plasma, and factor VIIa was measured after incubation for 30 minutes at 23°C as previously described [13]. The concentrations were chosen to bracket the concentrations used in the perfusion circuit in this study (18.4 µM). This concentration of aprotinin is equivalent to 1,200 KIU/mL, or 0.168 mg/mL. Factor VIIa inhibition from three separate experiments was analyzed.

Simulated extracorporeal circuit
Each perfusion circuit (surface area of 0.46 m²) was assembled from silastic tubing ( in. inside diameter [ID]) (Dow Corning, Midland, MI), polycarbonate connectors, a polyvinyl chloride venous reservoir bag (Gish Biomedica, Santa Ana, CA), and a 0.4-m² spiral/coil membrane oxygenator (model 0400-2A; Avecor, Inc, Plymouth, MN) [8]. The perfusion circuit and all labware contacting blood were heat sterilized. Human blood (300 mL) was drawn from healthy male donors (age 24–36 years) through a 16-gauge, 15-inch needle and polyvinyl tubing directly into a venous reservoir bag containing beef lung heparin (5 U/mL) blood (Upjohn Co, Kalamazoo, MI) and dextrose (2.25 mg/mL blood). Donors gave informed written consent and abstained from all medication for at least 2 weeks before donation. The protocol was approved by the Institutional Review Board of the University of Pennsylvania.

Each circuit was used once and discarded. Blood and gas compartments were flushed with 100% carbon dioxide for 15 minutes and primed from the circuit reservoir by vacuum. Aprotinin (1.4 mg/mL stock; Miles, Inc, West Haven, CT) (n = 7), or an identical volume of saline (n = 7) was added directly to reservoir blood and gently mixed before starting recirculation to give a final concentration of 18 µM. Blood was recirculated using a precisely shimmed, barely occlusive roller pump (Sarns, Ann Arbor, MI) at 0.29 L/min for 240 minutes. Oxygenators were ventilated with 95% oxygen-5% carbon dioxide at a rate of 0.7 L/min. Blood temperature was maintained at 28°C by immersing the reservoir bag in a constant-temperature water bath. One blood sample was drawn directly from the donor, and six blood samples (9 mL) were drawn from the reservoir into polypropylene tubes before recirculation (zero time) and 5, 30, 60, 120, 180, and 240 minutes after starting recirculation [8]. No samples for Mac-1 expression on mononuclear cells or tissue factor activity were taken at 5 and 30 minutes, because a previous study from our laboratory [14] showed no changes during that time interval. Similarly, no samples for kallikrein-C1-inhibitor complexes were taken at 180 and 240 minutes. Zero time and standing (uncirculated) control samples were obtained from the venous reservoir before connection to the circuit. The standing control sample (5 U/mL heparin, 2.25 mg/mL dextrose) was incubated for 4 hours at 28°C before processing. All blood, including standing control samples, was collected into acid-citrate-dextrose (9:1 by volume) and centrifuged at 500x g for 20 minutes to remove most cells. The supernatant plasma was centrifuged at 27,000g for 5 minutes at 23°C to prepare cell-free plasma, which was stored at -70°C for subsequent analyses.

Mononuclear cell separation
Mononuclear cells were prepared with slight modification of the method described earlier [14]. Whole blood was layered onto Ficoll-Paque (Pharmacia Biotech) and centrifuged 400g for 30 minutes at room temperature. For Mac-1 studies, cell separation was carried out at 4°C to avoid upregulation by temperature. Mononuclear cells were harvested from the interphase and washed twice in Hanks’ balanced salt solution without Ca²⁺ and Mg²⁺ (HBSS) containing 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY). Cell count was adjusted to 10⁶/mL for flow cytometry analysis and for assay of procoagulant activity (PCA) of intact viable cells. Viability was typically greater than 95% as measured by the Trypan blue exclusion test [14].

Cell staining for flow cytometry
Isolated mononuclear cells (10⁶/mL) were washed twice in 1 mL of HBSS containing 1% fetal bovine serum (washing buffer) and resuspended in mouse immunoglobulin (Ig)G (Sigma Chemical Co., St. Louis, MO) (20 mg/mL) to block nonspecific binding. Cells were incubated for 10 minutes, and then 10 mL of mouse monoclonal anti-human CD11b (Sigma) (4 mg/mL) and a sheep anti-mouse, IgG-FITC conjugate (Sigma) (4 mg/mL) were added. Cells were incubated for 30 minutes. After washing twice with buffer, cells were fixed overnight in 0.5% paraformaldehyde in HBSS. Cells were centrifuged at 400g for 10 minutes, washed twice in HBSS, and stored in dark until analysis.

Fluorescence was analyzed in an Ortho Cytofluorograf (Ortho Diagnostics, Inc, Westwood, MA) equipped with a model 2150 data-handling system. Fluorescence of polymorphoneutrophils (PMN), monocytes, and lymphocytes was determined by gating on each cell population as based on different forward and side scatters. Mean fluorescence of samples was compared with that of controls obtained from the venous reservoir before recirculation (baseline sample) and an unrecirculated standing control incubated at 28°C for 240 minutes in silicone rubber.

Procoagulant activity assay (PCA)
PCA of lysed mononuclear cells (10⁶/mL) was measured by a one-step recalcification time as described previously [15]. To quantify tissue factor (TF), serial dilutions of recombinant native human TF (T.S. Edgington, MD; Scripps, La Jolla, CA) were used to generate a standard curve. PCA was calculated in nanograms of TF/10⁶ mononuclear cells. The mononuclear cells were stimulated with a lipopolysaccharide as described [14], and the resulting TF concentration was set as 100% for each recirculation. TF was expressed as percent of this maximal stimulus. Previous studies have shown that PCA measured with this assay during simulated extracorporeal circulation was completely neutralized by anti-human TF antibody [14]. Factor VII levels in plasma were measured as described previously [15].

Other measurements
Concentrations of prothrombin fragments, F1.2 (Behring Diagnostics, Inc) and thrombin-antithrombin complex (TAT; Behring Diagnostics, Inc) were measured by enzyme-linked immunosorbent assay using commercial assay kits. Kallikrein-C1-inhibitor concentration was measured by a radioimmunoassay as previously described [16].

Statistical analysis
Data are expressed as mean ± SEM and were analyzed by two-way analysis of variance (ANOVA) (drug, time). The F statistic was calculated by two-way ANOVA with the Bonferroni correction for repeated measures (SPSS 7.5). All data for each group were analyzed by an unpaired t test with the Bonferroni correction compared with the donor sample. If a group effect was significant (p < 0.05), measurements between groups for specific times were compared by Student’s unpaired t statistic. Because of a significant difference (p = 0.05) in monocyte TF expression in donor blood (before aprotinin was added) between groups, each individual donor measurement of TF (expressed as percent of LPS stimulated value) was subtracted from all subsequent samples before statistical analysis using the two-way ANOVA. Probability values are expressed as exact numbers, eg, p = 0.01.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
In vitro study of plasma factor VIIa inhibition by aprotinin
Factor VIIa in the absence of aprotinin in pooled normal plasma is 0.43 ng/mL, similar to a previous study [15]. Detectable inhibition with aprotinin in vitro occurs at 36 µM and is proportional to concentration (Fig 1). The IC₅₀ is 55 µM; no inhibition occurs at 18.4 µM, the concentration used in the simulated bypass.

View larger version (14K): [in this window] [in a new window]   Fig 1. Effect of aprotinin concentration in vitro on the levels of factor VIIa in pooled normal plasma. Data are mean ± SEM of three separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig 2. Effect of aprotinin on F1.2 levels in simulated extracorporeal circulation. Bars, mean ± SEM; n = 7 in each group; open squares = control; closed circles = aprotinin.

View larger version (15K): [in this window] [in a new window]   Fig 3. Effect of aprotinin on monocyte Mac-1 expression in simulated extracorporeal circulation. Symbols are as in Figure 2.

View larger version (14K): [in this window] [in a new window]   Fig 4. Effect of aprotinin on monocyte tissue factor expression in simulated extracorporeal circulation. Symbols are as in Figure 2. The relative tissue factor activity (percent of the LPS-stimulated value) is corrected by subtracting the mean value in the donor sample from each time point and is expressed as % LPS-stimulated value.

View larger version (14K): [in this window] [in a new window]   Fig 5. Effect of aprotinin on kallikrein-C1-inhibitor complexes in stimulated extracorporeal circulation. Symbols are as in Figure 2. Data are mean ± SEM for five separate experiments based on previous experiments [12].

	Comment

Top Abstract Introduction Material and methods Results Comment References

In the absence of a wound, monocyte tissue factor expression increases after a delay of 2 hours [14]; thus, in this study, the early increase in thrombin markers in the control group is not explained by activation of monocyte TF or activation of factor VII. Inhibition by aprotinin of tissue factor expression contributes to a delayed increase in F1.2 and TAT. In clinical open heart surgery, however, the extrinsic coagulation pathway is activated within 30–45 minutes of starting CPB [15]. Addition of pericardial blood to the perfusate increases circulating thrombin [15].

Aprotinin partially inhibits thrombin formation and activity by inhibiting kallikrein. Partial inhibition of kallikrein attenuates the feedback activation of factor XII and thus decreases activation of factor XI to XIa. This mechanism explains the decreased thrombin formation by the intrinsic coagulation pathway, which is activated in both the simulated extracorporeal perfusion circuit and during open heart surgery. At concentrations used in CPB (4.2–8.5 µM, 274–540 KIU/mL), aprotinin inhibits kallikrein (Ki = 36 nM). This mechanism best explains the early inhibition of thrombin formation in the simulated perfusion circuit.

Aprotinin is a serine protease inhibitor that has both coagulant and anticoagulant properties. The primary target is plasmin [6], which aprotinin inhibits directly. In addition, aprotinin inhibits fibrin degradation by two other mechanisms. By inhibiting kallikrein, aprotinin reduces formation of bradykinin, which stimulates endothelial cells to release tissue plasminogen activator (t-PA). Furthermore, aprotinin attenuates release of neutrophil elastase, a powerful enzyme for degrading fibrin by inhibiting kallikrein, a potent neutrophil agonist [19].

Aprotinin also modulates fibrinolysis. Aprotinin inhibits the conversion of prourokinase to urokinase by plasmin. Aprotinin also inhibits activated protein C [20], a natural anticoagulant that proteolyzes and inactivates factors Va and VIIIa. Activated protein C combines stoichiometrically with plasminogen activator inhibitor [21] and, by depleting the inhibitor, increases fibrinolysis. Aprotinin may decrease fibrinolysis by inhibiting protein C; however, the Ki = 1,100 µM. Plasmin is not produced in the simulated circuit [22], but, in vivo, the net effect of aprotinin on protein C is procoagulant.

Aprotinin is procoagulant for platelets. Although aprotinin reduces alpha granule release during simulated extracorporeal circulation [8], the drug attenuates postoperative increases in bleeding time [1]. This improvement in overall platelet function is probably due to preventing internalization and proteolytic degradation of platelet GPIb/IX receptors caused by plasmin [23]. The antiplasmin effect on platelets overcomes the loss of ability of von Willebrand factor to cause platelet adhesion by binding to GPIb/IX.

In simulated extracorporeal circulation, monocytes provide the only source of tissue factor, and expression of tissue factor is delayed [14]. During clinical open heart surgery, the wound provides the major stimulus to the extrinsic coagulation pathway [15], and monocyte tissue factor expression, factor VII activation, and thrombin formation occur early [15]. Factor XIIa, activated complement, and probably other proteases involved in inflammation bind to and activate monocytes. By inhibiting kallikrein, aprotinin indirectly inhibits the formation of factor XIIa and inhibits activation of C1 of the classical complement pathway, and thus may suppress monocyte TF expression.

The wound is a major source of thrombin during open heart surgery, and protocols that wash or do not return blood aspirated from the wound reduce the thrombotic potential of the perfusate [24]. Topical use of aprotinin after CPB before the wound is closed reduces postoperative blood loss [5], and systemic aprotinin reduces the amount of fibrinolysis within the wound [25]. Blood conservation requirements for most operations preempt discarding wound blood; reinfusion of packed cells after washing (discarding plasma) is not always feasible. The discovery of the inhibitory effect of high doses of aprotinin on expression of monocyte tissue factor and the extrinsic coagulation pathway introduces the opportunity to suppress thrombin generation by local instillation of aprotinin directly into the wound (instead of the perfusion circuit) during the procedure. This practice, in combination with low-dose systemic aprotinin [25], may enhance suppression of thrombin generation without compromising inhibition of fibrinolysis during complex operations in which washing wound blood is not feasible.

	Acknowledgments

 
The excellent technical assistance of Dr Ling Sun is acknowledged and appreciated. The manuscript and figures were skillfully prepared by Rita Stewart. This work was supported by Grant HL-47186 from the National Heart, Lung, and Blood Institute, National Institutes of Health.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): L. Henry Edmunds, Jr Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Khan, M. M.H. Articles by Colman, R. W. Search for Related Content PubMed PubMed Citation Articles by Khan, M. M.H. Articles by Colman, R. W.