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Ann Thorac Surg 1996;61:1188-1193
© 1996 The Society of Thoracic Surgeons
Departments of Thoracic and Cardiovascular Surgery and Pathology, Loyola University Medical Center, Maywood, Illinois
Accepted for publication December 13, 1995.
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentReferences |
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Methods. To study the cytoprotective effect on platelets, we collected blood samples from patients during cardiopulmonary bypass (CPB) operations performed with or without aprotinin. Analysis included whole-blood flow cytometry.
Results. The highest percentages of activated platelets (positive for GMP-140 expression) were bound to leukocytes and erythrocytes in all CPB patients. Platelet-platelet activation did not reveal any marked differences between groups. However, in the platelet-cell bound region, increased ristocetin-stimulated platelet activation was observed from 30 minutes on CPB to 90 minutes after CPB with aprotinin (11.9% ± 5.1% to 33.1% ± 8.6%; p< 0.05), but not without aprotinin (17.5% ± 0.1% to 17.9% ± 2.3%). Platelet autoactivation increased more in the untreated group with time on CPB.
Conclusions. This study demonstrates that in the presence of aprotinin, platelets remain unstimulated during CPB and the von Willebrand GPIb-mediated activatability of platelets is preserved, thus maintaining a viable platelet population. Most important, this study reveals that these mechanisms are more related to platelet-leukocyte than to platelet-platelet interactions.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Aprotinin (Trasylol; Bayer, West Haven, CT) is a proteinase inhibitor of plasmin, kallikrein, thrombin, trypsin, and chymotrypsin. Aprotinin reduces fibrinolytic activation and thrombin generation during operations [4]. Previous studies have shown that aprotinin reduces postoperative blood loss by up to 50% [5,6]. However, the described effects of aprotinin alone do not seem to account for the significant decrease in postoperative blood loss, as clinical studies often lack significant findings of these indices between control and aprotinin-treated groups.
Current theories propose that aprotinin has a direct or indirect cytoprotective effect on platelets by preventing the cellular damage that occurs during the ``first pass'' through the CPB circuit [5,7,8]. One of the most likely mechanisms shown to date is the inhibition of plasmin by aprotinin, which then eliminates the suppression of platelet function by plasmin [1,7,9–11]. However, this indirect effect and other proposed mechanisms have not been reproduced in all experimental studies and remain unconfirmed in the clinical state.
We designed a study to evaluate platelet activation in patients having cardiac operations and receiving aprotinin. Previous in vitro studies were unable to mimic appropriately the operative setting, and clinical studies have used classic platelet assays, which lacked sensitivity and specificity. For our study, we chose an optimized flow cytometric method to study platelets in whole blood (Koza MJ, Walenga JM, Bermes EW Jr, Pifarré R, Fareed J, Shankey TV; unpublished results) using blood samples collected during operation and analyzed immediately. A monoclonal antibody to the GPIIIa receptor (CD61), a platelet surface marker expressed on both activated and nonactivated platelets, was used to identify platelets. We simultaneously analyzed a second platelet marker, GMP-140 (CD62), expressed on the surface of activated platelets as the internal alpha-granule membrane becomes integrated with the cell surface membrane. Using the flow cytometer, size and granularity measurements of the platelet could be performed in addition to detection of the CD61 and CD62 antibodies. Thus, we evaluated platelet microparticle formation, aggregation, and adhesion in whole blood.
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Material and Methods |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Aprotinin administration was begun after induction of anesthesia and was continued until the patient was transferred to the intensive care unit. Aprotinin was administered as a bolus plus infusion and pump prime at a total dose of 4.5 million KIU (630 mg) (n = 7). A control group included patients (n = 10) who underwent operation without the use of aprotinin.
Sample Preparation
Blood was collected from the patients at four separate times: (1) after induction of anesthesia, before initiation of thoracotomy, and before aprotinin was given; (2) 30 minutes after the start of CPB; (3) at the end of CPB after protamine sulfate administration (at a heparin concentration of zero); and (4) 90 minutes after CPB. Blood was collected through a Swan-Ganz catheter (sample 1) or through the CPB pump (samples 2, 3, and 4), as these access sites for blood collection were readily available. This sampling allowed optimal analysis of the effect of the CPB circuit on the blood. After collection, 1 mL of patient whole blood (with no additional anticoagulant agent) was incubated with either 100 µL of saline solution or with the platelet agonist adenosine diphosphate (ADP) (2.5 x 10-6 mol/L final concentration) (Sigma, St. Louis, MO) or ristocetin (1.5 mg/mL final concentration) (Biodata, Horsham, PA) at 37°C. A second milliliter of whole blood was placed in a tube containing ethylenediamine tetraacetic acid for blood cell count.
A flow cytometric method previously described [12] was used for this study. Briefly, a 50-µL aliquot of each sample was fixed with 1 mL of cold, filtered 1% paraformaldehyde either immediately (baseline activation, saline solution) or after a 2.5-minute (ADP) or 5.0-minute (ristocetin) incubation period at 37°C. Previous studies have revealed these to be optimal in vitro activation times for these agonists or saline solution. After fixation, each sample was incubated at 4°C for 30 minutes and then centrifuged at 600 g for 10 minutes. The fixative was removed, and the samples were resuspended in 400 µL of Tyrode's buffer.
A 100-µL aliquot of each sample was incubated with 100 µL of a 1:50 dilution of CD61-FITC monoclonal antibody (anti-GPIIIa labeled with fluorescein isothiocyanate) (Becton Dickinson, San Jose, CA) and 100 µL of 1:50 CD62-PE monoclonal antibody (anti–GMP-140 labeled with phycoerythrin) (Becton Dickinson). The labeled samples were incubated in the dark for 30 minutes at room temperature to allow antibody binding, and then an additional 400 µL of Tyrode's buffer was added to dilute the sample.
Flow Cytometric Analysis
Flow cytometric analysis of monoclonal antibody (CD61 plus CD62)-stained platelets in whole blood was performed using an Epics-XL flow cytometer (Coulter Corp, Hialeah, FL). All samples were analyzed at the slowest flow rate (approximately 10 µL/min). For all samples, list mode files were saved from a minimum of 50,000 gated events, including signals collected on each cell for forward angle light scatter (FALS), side scattered light, and FITC and phycoerythrin fluorescence. Fluorescence of FITC was collected through the 525 ± 10–nm bandpass filter, whereas phycoerythrin fluorescence was collected using a 600-nm long-pass dichroic filter and a 575 ± 10–nm bandpass filter. All signals were acquired using logarithmic amplification (on a four decade log scale). Samples from each individual patient were initially analyzed to demonstrate that greater than 90% of the platelets were labeled with CD61 [11]. Subsequent samples were collected using an FITC threshold discriminator set to exclude all events not labeled with the platelet-specific antibody (CD61-FITC). The relative size of particles passing through the flow cell was determined using the FALS signal obtained from standardized beads of known size (1 to 10 µm).
Analysis of list mode data files was performed using MPlus AV software (Phoenix Flow Systems, San Diego, CA). Histograms were constructed from CD61 versus FALS to define the percentage of labeled platelets in three different categories. As shown in Figure 1
, These include platelet microparticles (region A), defined as particles with platelet surface antigens (CD61) and lacking FALS (size); platelet-platelet aggregates (region B); and platelet–white blood cell (WBC) or platelet–red blood cell (RBC) complexes (region C). Very low levels of platelet microparticles were observed in unstimulated platelet preparations (see Fig 1A), whereas a significant increase in this population was observed after in vitro agonist (ADP)-induced activation (see Fig 1B). Similarly, significant increases were seen in the platelet-platelet aggregates; based on size estimates from standardized beads, these aggregates increased in size from 1 to 5 µm, to more than 10 µm (region B; see Fig 1b). The numbers of platelet-WBC and -RBC complexes also increased significantly after platelet activation (see Fig 1; region C).
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). Using the MPlus AV software, we constructed histograms of CD62 (platelet activation) versus CD61. As shown in Figure 2A, quadrants were first determined on unstimulated, baseline samples (sample taken after induction of anesthesia and immediately fixed in paraformaldehyde) and setting an arbitrary value of approximately 2% CD62-positive platelets equal to 0% platelet activation. Figure 2B shows the increased CD62 expression, on platelets bound to cells other than platelets, after ADP stimulation. Using list mode data analysis, we calculated the percentage of activated (CD62-positive) platelets for each platelet region. Because the percentage of activated platelets was calculated for each platelet population (based on the same total number of platelets), changes in actual platelet counts would not affect the analysis of the platelet populations.
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Results |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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. Patients receiving aprotinin demonstrated a trend of lower postoperative blood loss compared with those patients who did not receive aprotinin. Blood collected from the chest drainage tubes 24 hours after operation was 7.7 ± 1.1 mL/kg in the control group and 5.2 ± 0.9 mL/kg in the aprotinin-treated group. This difference was not statistically significant.
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In the patients having cardiac operations, platelet autoactivation was determined; i.e., the activated state of circulating platelets was measured without in vitro stimulation by a platelet agonist. Evaluation of all platelet events (whole population) showed no significant activity (GMP-140 expression) in the untreated or the aprotinin group during operation (1.0% ± 0.8% CD62) or after operation (0.8% ± 0.6% CD62) (Fig 3). In the subpopulation of platelets bound to RBCs or WBCs (region C; see Fig 1), autoactivation was present in both the aprotinin (2.9% ± 0.5% CD62) and untreated groups (4.5% ± 1.6% CD62) during operation. Platelet activity was therefore more enhanced in the nontreated group. At the termination of aprotinin administration, platelet autoactivation was still more enhanced in the untreated group (8.1% ± 2.7% CD62) compared with the aprotinin group (4.0% ± 1.2% CD62), as measured in the platelets bound to RBCs or WBCs.
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). Platelet activation evaluated for all platelet events showed no significant difference between the aprotinin versus control group at any of the four perioperative times. When only platelets bound to RBCs or WBCs were evaluated, a higher level of activity was observed than for the entire platelet population (average 21% CD62 versus 8%), but again no difference between the groups was observed. Over the perioperative period, the aprotinin group ranged from 22.4% ± 4.3% to 13.6% ± 1.9% CD62 after ADP stimulation, and the control group ranged from 21.3% ± 3.2% to 15.1% ± 1.6% CD62 in the platelet-cell bound region. The normal range for our method in normal individuals is 20% to 25% CD62 expression after ADP stimulation.
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). However, with aprotinin only and only in the subpopulation of platelets bound to RBCs or WBCs, increasing platelet activation was observed from 30 minutes on CPB to 90 minutes after CPB (11.9% ± 5.1% to 33.1% ± 8.6% CD62; p < 0.05). This increased activity was not observed in the control group (17.4% ± 0.1% to 17.9% ± 2.3% CD62) or in the platelet-platelet or platelet-fragment region for either group at any time. The normal range for our method in normal individuals is 25% to 35% CD62 expression after ristocetin stimulation.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Studies have suggested that during the ``first pass'' through the CPB circuit, platelets show increased activation (thromboxane B2 release), and then show increased expression of GMP-140 after CPB. In the presence of aprotinin, however, the platelet receptors GPIIb/IIIa and GPIb, which are normally hydrolyzed, damaged, or internalized, are conserved [5, 7, 8]. These studies have been of limited size and have often used nonclinical, unconventional techniques, such as electron microscopy to detect platelet aggregation and radioisotopes to detect platelet receptor expression.
Our present study differs from previous studies in that flow cytometry was used in a surgically relevant setting. Flow cytometry is a newly developed technique for simultaneously assessing platelet activation and aggregation that can identify and quantitate discrete platelet populations (single platelets, platelets bound to other cells, and platelet fragments). It holds several advantages over the classic techniques: whole-blood analysis eliminates the need for multiple washing and centrifugation steps that can lead to artifactual platelet activation or damage; it is a sensitive and specific assay providing information on both platelet activation and aggregation; it allows observation of cell to cell interactions; and, most important, flow cytometry allows the enumeration of different platelet populations (Koza MJ; unpublished results) [13, 14]. This technique, which we have optimized for whole-blood analysis of platelets, allows complete platelet analysis in the most physiologic manner possible.
An important observation from this study is that the differences in the platelet activities between the aprotinin and untreated groups were most apparent in the platelet-cell bound (leukocytes, erythrocytes) region of analysis. Through the use of flow cytometry, it has been revealed that the most activated platelets at any perioperative time in cardiac operations are a subpopulation of activated platelets that are bound to other cells. There were less marked functional differences in platelets bound to platelets (platelet aggregation) or in platelet microvesicle formation between the treatment groups. This supports our own earlier data and those of other investigators, in which the effects on platelet aggregation by aprotinin could not be demonstrated because whole blood was not used [11].
By flow cytometric analysis, resting platelets (autoactivation) showed higher activity levels only in the untreated group, particularly in the platelet-cell bound region. Activatability of platelets by ADP did not differ between the aprotinin-treated and untreated group at any time. There was, however, a loss of platelet activation by the end of CPB for both groups. This loss of function has been confirmed in our laboratory by platelet aggregation assay (data not shown).
In contrast, ristocetin-stimulated platelets (von Willebrand factor mediated) in aprotinin-treated patients demonstrated more activatability than in the control group at the end of CPB and 90 minutes after CPB. This was observed only in the platelet-cell bound region. Thus, these data suggest that the effect of aprotinin is directed toward the GPIb receptor and not through the mechanism by which ADP activates the platelet. In a related study, we evaluated patient whole-blood samples immediately before and after protamine was administered during operation. Platelet activation results from 6 patients undergoing CPB, analyzed by flow cytometry as described herein, demonstrated that the protamine given at the end of operation was not the cause of the observed platelet effects, as CD62 expression was the same at both times (unpublished data).
This study confirms in a clinical population an earlier described mechanism of action of aprotinin, ie, GPIb modulation of platelet function. This study further reveals, however, that this interaction is not necessarily platelet-structure or platelet-platelet in nature but, more important, involves a platelet-leukocyte/erythrocyte (most likely platelet-leukocyte) interactive mechanism. This implies an association between the antiinflammatory and hemostatic activation effects of aprotinin in reducing postoperative bleeding.
The association between leukocytes and hemostasis is not typically made, although limited studies, particularly on tissue factor, have suggested several lines of evidence linking these two components. Platelet release promotes leukocyte adherence in vitro, and leukocytes can stimulate platelet activation. Leukocytes also induce thrombosis. The exact role that leukocytes play is only beginning to be determined.
Maugeri and associates [15] showed that leukocyte-derived cathepsin G induces the expression of GMP-140 on platelet surfaces. This finding correlates with our data, which showed decreased GMP-140 in circulating platelets (autoactivation) of the platelet-cell bound region in the presence of aprotinin. Solum and colleagues [16] suggested that complement degrades platelet membranes in a manner that reduces the ristocetin-induced activation but not adenosine triphosphate release, and that this effect was targeted toward an action-binding protein linking GPIb and the submembranous cytoskeleton of the platelet. This also correlates with our data, in which the ristocetin-induced platelet activation response (not the ADP-induced response) was maintained in the presence of aprotinin when the platelet-cell bound region was evaluated.
In conclusion, this study has strengthened the data that support aprotinin modulation of platelet activation through the GPIb receptor during CPB. With aprotinin, the ability of the platelet to respond to shear forces and foreign material (shown by ristocetin stimulation) is maintained, thus manifesting as reduced postoperative blood loss. Thus, when a platelet's responsiveness, which we measured by the presence of activatable platelet receptors (GPIb), is maintained postoperatively, the platelet is able to contribute to hemostasis and decrease the amount of blood loss, whereas a platelet that is present yet nonreactive (without activatable platelet receptors) is unable to respond to its agonist and cannot support adequate hemostasis. The mechanism of ADP-induced aggregation, being different from the adhesion induced by ristocetin, is not affected by aprotinin.
Our study, moreover, demonstrates that the effect of aprotinin is mediated through the platelet-leukocyte adhesion/interaction. Although the many interactions between platelets and leukocytes require further investigation as to their function, we do know that one of these interactions involves the reversible binding of leukocytes to the platelet through the GMP-140 receptor. This particular mechanism may be key to finally understanding the overall mechanism of action of aprotinin, associatingthe hemostatic and the antiinflammatory interactions of aprotinin on platelets.
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Footnotes |
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References |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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