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CARDIOVASCULAR ANESTHESIA

The Effects of Aprotinin on Platelets In Vitro Using Whole Blood Flow Cytometry

Sibylle A. Kozek-Langenecker, MD^*, S. Fazal Mohammad, PhD^,, Takahisa Masaki, PhD^§, Wayne Green, PhD^||, Craig Kamerath^¶, and Alfred K. Cheung, MD^§,¶

*Department of Anesthesiology, University of Vienna, School of Medicine, Vienna, Austria; Department of Pathology, Artificial Heart Laboratory, §Division of Nephrology & Hypertension, Department of Medicine, ||Huntsman Cancer Institute, University of Utah School of Medicine; and ¶Medical and Research Service, Veterans Affairs Medical Center, Salt Lake City, Utah

Address correspondence and reprint requests to Sibylle A. Kozek-Langenecker, MD, Department of Anesthesiology and General Intensive Care, University of Vienna, Währinger Gürtel 18-20, 1090-Vienna, Austria. Address e-mail to sibylle.kozek-langenecker @univie.ac.at.

	Abstract

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We sought to evaluate the effects of aprotinin on the number and function of the platelet glycoprotein (GP) IIb–IIIa receptor and on the expression of P-selectin in vitro in order to gain insight into the potential mechanisms involved in the platelet-protective action of aprotinin during cardiopulmonary bypass. Aprotinin at 50 to 200 kallikrein inhibiting units/mL decreased the expression of activated GP IIb–IIIa complex in response to adenosine diphosphate or thrombin receptor activator peptide 6 in a dose-dependent manner in both citrated and heparinized whole blood experiments. Aprotinin inhibited adenosine diphosphate-induced platelet aggregation, but it exhibited no effect on the expression of GP IIIa and P-selectin. These results indicate that aprotinin interferes with the platelet fibrinogen receptor function during pharmacological activation. Reduced aggregability and platelet adhesion to fibrinogen adsorbed to synthetic surfaces in the presence of aprotinin may prevent platelet consumption during clinical cardiopulmonary bypass. This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated GP IIb–IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces.

Implications: This in vitro study demonstrates that aprotinin decreases the agonist-induced expression of activated glycoprotein IIb–IIIa receptors that play a major role in platelet aggregation and adhesion to biomaterial surfaces.

	Introduction

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Platelet dysfunction is considered to be the most frequent cause of postoperative blood loss after cardiopulmonary bypass (CPB) (1). Aprotinin reduces postoperative bleeding after cardiac surgery (2). Although the mechanism of this beneficial effect has not been fully delineated, it appears that a platelet-protective effect during extracorporeal circulation plays a role (2–8).

During extracorporeal circulation, platelets are activated by high shear rates and biologically active products that are released, such as adenosine diphosphate (ADP), thrombin, thromboxane A₂, and proteolytic enzymes (9). Platelet activation transforms platelet membrane glycoprotein (GP) IIb–IIIa complexes on the platelet surface to a conformational state competent for binding fibrinogen (10). This reaction is a prerequisite for platelet aggregation and interaction of activated platelets with fibrinogen adsorbed to artificial surfaces (11). Platelet adhesion to the extracorporeal circuit promotes further platelet activation and consumption (12). Although it has not been substantiated experimentally, some authors have postulated a protective effect of aprotinin on the function of platelet fibrinogen receptor during clinical CPB (6,7). In contrast, Wahba et al. (13) found no effect of aprotinin on the number of platelet GP IIb–IIIa receptors during clinical CPB. A potential drawback of the study by Wahba et al. (13) was that an activation-independent antibody against GP IIb was used for flow cytometric analysis, which permitted assessment of the number only and not of the functional state of GP IIb–IIIa (14). Because neoepitopes on activated GP IIb–IIIa complexes can be detected by flow cytometry by using activation-dependent antibodies such as PAC-1 (15), we used both an activation-dependent and an activation-independent antibody to GP IIb–IIIa in order to investigate the effects of aprotinin on the expression and function of the platelet fibrinogen receptor. The effect of aprotinin on platelet-release reaction was also evaluated. P-selectin, which is expressed on the surface of activated platelets as the internal alpha granule membrane becomes integrated into the cytoplasmatic membrane (16), served as a marker for platelet secretion. At present, the method of whole blood flow cytometry permits assessment of platelet activation in the most physiological manner possible (14,15). We used this method for platelet analysis in resting condition and after exposure to a weak platelet agonist, ADP, or a strong agonist, thrombin receptor activator peptide 6 (TRAP).

	Methods

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We studied the blood obtained from 20 healthy adult volunteers. All participants denied taking any medication known to alter platelet function within the previous 14 days. The study was approved by our institutional review board.

Flow Cytometric Analysis
Blood was drawn by venipuncture without stasis from the antecubital vein into Vacutainer® tubes (Becton Dickinson, Rutherford, NJ) containing 3.2% trisodium citrate or into plastic tubes containing unfractionated porcine heparin at final concentrations of 0, 0.5, or 5 IU/mL and then diluted (1:5) in phosphate-buffered saline (100 mM sodium phosphate, pH 7.3, and 0.145 M NaCl). Citrated samples were incubated with aprotinin at final concentrations of 0, 50, or 200 kallikrein inhibiting units (KIU)/mL at 37°C for 5 min without agitation. Heparinized samples were incubated with aprotinin at 0 or 200 KIU/mL. Samples incubated with phosphate-buffered saline served as a control. Each test sample was divided into three aliquots: the first aliquot was incubated without a platelet agonist (resting condition); the second aliquot was incubated with a weak agonist ADP (10 µM) and the third aliquot was incubated with a strong agonist TRAP (60 µM) at 37°C for 5 min without agitation. The response to in vitro agonist stimulation allowed assessment of hemostatic properties of platelets. The response to ADP was determined because this mediator is released during platelet activation in vivo (9) and because ADP is a well accepted platelet agonist for in vitro platelet assays (15). The response to TRAP was evaluated because it mimics the effects of thrombin (17), which is generated at sites of contact with artificial surfaces (18).

Each test sample was further divided into two aliquots for fluorescent staining. Flow cytometric analysis was performed to determine the expression of activated GP IIb–IIIa complex, GP IIIa, and P-selectin as described previously (19). Briefly, to determine the expression of activated GP IIb–IIIa complex, one aliquot of each sample was incubated with a fluorescein isothiocyanate (FITC)-conjugated activation-dependent antihuman platelet GP IIb–IIIa monoclonal antibody (PAC-1) (14). The number of GP IIb–IIIa receptor sites was determined by incubating one aliquot of each sample with a FITC-conjugated antihuman platelet GP IIIa monoclonal antibody (anti-CD61), that reacts with a determinant common to unactivated and activated GP IIb–IIIa. The percentage of platelets expressing P-selectin was determined by incubating a third aliquot simultaneously with phycoerythrin-conjugated monoclonal antibody against human platelet P-selectin (anti-CD62P) and a FITC-conjugated activation-independent antihuman platelet GP IIIa monoclonal antibody (anti-CD61). After a 30-min incubation with saturating concentrations of the monoclonal antibodies at room temperature in the dark, samples were fixed in 1% paraformaldehyde (pH 7.3) at 4°C.

In each experiment, one sample was stained with FITC-conjugated, isotype-matched, nonspecific mouse immunoglobulins as a negative control. Another sample, without the addition of conjugated antibody, served as an autofluorescence control. Fluorescence was measured with a FACScan flow cytometer and analyzed with CellQuest 3.1 software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Quantum fluorescence microbeads (Calibrite Beads; Becton Dickinson Immunocytometry Systems) were used each day for standardization of instrument settings.

Platelet Preparations for Platelet Aggregation Studies
Platelet-rich plasma (PRP) was obtained by centrifugation of citrated whole blood at 100 x g for 15 min, and platelet-poor plasma was prepared by centrifugation of the remaining blood at 1000 x g for an additional 10 min at 22°C. Platelet counts in the suspension medium were adjusted to a final concentration of 2.5 x 105/µL by mixing appropriate ratios of PRP and homologous platelet-poor plasma. Gel-filtered platelets (GFP) were prepared from PRP by a modification of the procedure described previously (19,20).

Measurement of Platelet Aggregation
Aggregation of platelets in GFP was induced by incremental concentrations of ADP at 37°C in separate tubes containing a reaction volume of 500 µL. Platelet aggregation was recorded as an increase in light transmission after the addition of ADP in a dual-sample aggregometer (DP-247F; Sienco Inc., Morrison, CO). Aggregation was quantified by the area under the aggregation curve from 0 to 3 min. To investigate the effect of aprotinin on platelet aggregation, GFP were incubated with aprotinin at final concentrations of 100 or 200 KIU/mL for 5 min at 37°C while GFP diluted in saline served as controls.

A power analysis reveals that a sample size of 10 would provide a power of >80% in detecting a difference in ADP-induced PAC-1 binding of at least 1 SD in citrated samples incubated with and without aprotinin 200 KIU/mL. Data were expressed as mean ± SD. Citrated blood samples were analyzed using analysis of variance for repeated measures. Post hoc comparisons between control and aprotinin exposures were made with Student’s t-test for paired data. The level of significance was adjusted according to Bonferroni’s correction. Heparinized blood samples with and without aprotinin exposure were analyzed using Student’s t-test for paired data. A two-tailed P < 0.05 was considered statistically significant.

	Results

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Evaluation of platelet surface GP IIb–IIIa in citrated whole blood is shown in Figure 1. The monoclonal antibody PAC-1 was used to identify activated GP IIb–IIIa receptors on platelets (Fig. 1A). As expected, resting platelets expressed a negligible number of activated GP IIb–IIIa complex, while activation with ADP or TRAP increased the expression of activated GP IIb–IIIa. Agonist-activated control samples without aprotinin are arbitrarily defined as 100%. Aprotinin attenuated the increase in GP IIb–IIIa expression on both ADP- and TRAP-activated platelets in a dose-dependent manner. At an aprotinin concentration of 200 KIU/mL, GP IIb–IIIa expression on ADP-activated platelets and on TRAP-activated platelets was reduced by 22% ± 4% (P < 0.05) and 27% ± 8% (P < 0.05), respectively.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of aprotinin on GP IIb–IIIa on resting, ADP-activated, and TRAP-activated platelets in citrated whole blood. Agonist-activated control samples without aprotinin are arbitrarily defined as 100%. A, Binding of PAC-1 to activated GP IIb–IIIa complex. Aprotinin inhibited the increase in GP IIb–IIIa expression on both ADP- and TRAP-activated platelets. Values are expressed as mean ± SD of 10 determinations for each condition performed in duplicate. * P < 0.05 versus control. B, Binding of anti-CD61 to GP IIIa. Aprotinin had no effect on the increase in GP IIIa expression on either ADP- or TRAP-activated platelets. Values are mean ± SD of 10 determinations for each condition performed in duplicate. GP = glycoprotein, ADP = adenosine diphosphate, TRAP = thrombin receptor activator peptide, PAC-1 = activation-dependent anti-GP IIb–IIIa monoclonal antibody, anti-CD61 = anti GP IIIa monoclonal antibody.

To further evaluate the observed effect of decreased expression of the fibrinogen binding sites on agonist-stimulated platelets in the presence of aprotinin (Fig. 1A), we studied platelet aggregation in response to the minimal dose of ADP required to produce maximal aggregation. Aprotinin decreased platelet aggregation in a dose-dependent manner (aprotinin 100 KIU/mL: 11% ± 3%, P < 0.05; aprotinin 200 KIU/mL: 20% ± 5%, P < 0.05 compared with control).

Evaluation of the expression of platelet P-selectin in citrated whole blood is shown in Figure 2. Platelets were identified as positive for GP IIIa antigen (CD61), and the presence of a phycoerythrin-labeled antibody against P-selectin (anti-CD62P) was used to determine the percentage of platelets expressing P-selectin. As expected, resting platelets negligibly expressed P-selectin, while activation with ADP or TRAP increased P-selectin expression significantly. The strong agonist TRAP increased P-selectin expression more than the weak agonist ADP. Aprotinin had no effect on the increased binding of anti-CD62P to P-selectin on both ADP- and TRAP-activated platelets.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of aprotinin on P-selectin expression on resting, ADP-activated, and TRAP-activated platelets in citrated whole blood. Aprotinin had no effect on the increased binding of anti-CD62P to P-selectin on either ADP- or TRAP-activated platelets. Values are expressed as mean ± SD of 10 determinations for each condition performed in duplicate. ADP = adenosine diphosphate, TRAP = thrombin receptor activator peptide, anti-CD62P = monoclonal anti-P-selectin antibody.

The effect of aprotinin on platelets in whole blood samples anticoagulated with unfractionated heparin is shown in Figure 3. Heparin increased the binding of PAC-1 on the surface of platelets activated by ADP or TRAP in a dose-dependent manner (Fig. 3A). Preincubation of heparinized samples with 200 KIU/mL aprotinin before agonist-activation reduced the increase in GP IIb–IIIa expression on both ADP- and TRAP-activated platelets. At a heparin concentration of 0.5 IU/mL, GP IIb–IIIa expression on ADP-activated platelets and on TRAP-activated platelets was reduced by 22% ± 15% (P < 0.05) and 39% ± 17% (P < 0.05), respectively. At a heparin concentration of 5 IU/mL, GP IIb–IIIa expression on ADP-activated platelets and on TRAP-activated platelets was reduced by 11% ± 10% and 13% ± 5% (P < 0.05), respectively.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of aprotinin on GP IIb–IIIa on resting, ADP-activated, and TRAP-activated platelets in heparinized whole blood. Agonist-activated control samples without heparin and aprotinin are arbitrarily defined as 100%. A, Binding of PAC-1 to activated GP IIb–IIIa complex. Aprotinin inhibited the increase in GP IIb–IIIa expression on both ADP- or TRAP-activated platelets. Values are expressed as mean ± SD of 10 determinations for each condition performed in duplicate. * P < 0.05 versus control. B, Binding of anti-CD62P to P-selectin. Aprotinin had no effect on the increased binding of anti-CD62P to P-selectin on both ADP- and TRAP-activated platelets. Values are mean ± SD of 5 determinations for each condition performed in duplicate. GP = glycoprotein, ADP = adenosine diphosphate, TRAP = thrombin receptor activator peptide, PAC-1 = activation-dependent anti-GP IIb–IIIa monoclonal antibody, anti-CD62 = monoclonal anti-P-selectin antibody.

	Discussion

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Aprotinin has been reported to preserve platelets during extracorporeal circulation (2–8). However, ex vivo studies on the mechanism by which aprotinin affects platelet function have yielded controversial results.These inconsistencies may reflect the variability in study designs and methods used for assessing platelet function or platelet receptors. We have reported that platelet aggregation is strongly related to the availability of activated GP IIb–IIIa complexes on the platelet surface (19). Therefore, we evaluated GP IIb–IIIa in response to agonist stimulation using a standardized experimental in vitro study design to gain insight into the potential mechanisms by which aprotinin may offer protective effects. This study confirms that the total number of GP IIb–IIIa complexes expressed on stimulation is unaffected by aprotinin, as determined by the unchanged binding of an activation-independent monoclonal antibody against platelet GP IIIa (anti-CD61; Fig. 1B) (3,13). However, results presented in Figure 1A and Figure 3A clearly demonstrate that aprotinin decreases agonist-induced availability of activated GP IIb–IIIa receptors competent for binding fibrinogen, as determined by the decreased binding of an activation-dependent antihuman platelet GP IIb–IIIa monoclonal antibody (PAC-1). Confirming previous reports (21), we also found impaired aggregation of isolated platelets in the presence of aprotinin. Together, these results indicate that aprotinin interferes with GP IIb–IIIa receptor function during pharmacological platelet activation. Decreased expression of fibrinogen binding sites on activated platelets could be caused by a direct platelet-inhibiting effect of aprotinin. If so, downstream platelet responses, such as release reaction, should be decreased similarly. However, alpha granule membrane protein expression on the platelet plasma membrane was unaffected by aprotinin, as determined by the unchanged binding of a monoclonal antibody to P-selectin (anti-CD62P; Figs. 2 and 3B). Comparable results of unchanged generation of ß-thromboglobulin, thromboxane A₂, and P-selectin expression in the presence and absence of aprotinin have been observed during clinical CPB (4,8,13,22,23). Another possible explanation for the reduced availability of fibrinogen binding sites on activated platelets could be a modification of the platelet cytoplasmatic membrane structure by aprotinin, which allows upregulation but not conformational activation of the GP IIb–IIIa complex after subsequent stimulation. This possible mechanism needs further evaluation.

For platelet analysis, sodium citrate is currently the most frequently used anticoagulant (15); however, unfractionated heparin is routinely used for anticoagulation in clinical CPB. Because both anticoagulants have been shown to influence platelet reactivity (24), which may alter the effects of aprotinin, we performed functional assays in both anticoagulants. Heparin, at concentrations used during clinical CPB (0.5 to 5 IU/mL), appeared to preactivate platelets for an increased response to further agonist stimulation (Fig. 3). This is consistent with previous reports that documented a potentiation of platelet activation by unfractionated heparin (25). The present study clearly shows that aprotinin reduces the availability of fibrinogen binding sites on activated platelets not only in standardized test media for coagulation assays containing citrate, but, most importantly, also in heparinized whole blood specimen (Figs. 1A and 3A).

One limitation of our study is that platelet agonist exposure is not equivalent to the multifactorial influences on platelets induced by clinical CPB (9). Although extrapolations from experimental conditions to complex clinical settings should be made with care, the present data indicate that the observed effect of aprotinin on platelet aggregability should be taken into consideration when investigating coagulation during CPB. Reduced aggregation and platelet adhesin to artificial matrices by aprotinin may prevent loss and consumption of platelets during CPB, thus maintaining a viable platelet population that is able to contribute to hemostasis manifesting in reduced postoperative blood loss.

	Acknowledgments

 
This work was supported in part by a grant from the Max Kade Foundation, New York, NY, to SK.

	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 Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Kozek-Langenecker, S. A. Articles by Cheung, A. K. Search for Related Content PubMed PubMed Citation Articles by Kozek-Langenecker, S. A. Articles by Cheung, A. K.