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Ann Thorac Surg 1998;65:691-695
© 1998 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Platelet and Neutrophil Activation During Cardiac Surgical Procedures: Impact of Cardiopulmonary Bypass

Center for Experimental Therapeutics and Reperfusion Injury Department of Anesthesia Research Laboratories, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
Department of Cardiac Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication September 3, 1997.

Dr Magnani, Coagulation Research Laboratory, Department of Anesthesia Research Laboratories, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115 (e-mail: magnani@zeus.bwh.harvard.edu).

	Abstract

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Background. Platelet and neutrophil activation plays a crucial role in reperfusion injury. To determine whether platelet and neutrophil activation occurs in the coronary circulation after cold cardioplegic arrest and reperfusion, we studied 22 patients undergoing coronary artery bypass or valve procedures, or both procedures.

Methods. Blood was sampled from the coronary sinus and the radial artery (A) before bypass; (B) immediately after cross-clamp release; and (C) 5 minutes after cross-clamp release; and was analyzed for surface markers of platelet (CD62P) and neutrophil (CD11b) activation.

Results. During bypass, platelet activation increased significantly (p < 0.01) over prebypass values, but no difference was seen between arterial and coronary sinus samples. Neutrophil activation also increased significantly (p < 0.001) during bypass, but there was no difference between arterial and coronary sinus samples.

Conclusions. Cellular activation occurs locally in the coronary circulation during bypass, but no more so after cold cardioplegic arrest and reperfusion.

	Introduction

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Activated platelets and neutrophils are central to the reperfusion syndrome seen after acute coronary occlusion syndromes such as unstable angina and myocardial infarction. Activated platelets release vasoconstrictors such as ADP, serotonin, and thromboxane A₂ [1], whereas activated neutrophils produce oxygen-free radicals and other cytotoxic substances known to cause endothelial dysfunction, myocardial injury, and vascular edema (the "no-reflow" phenomenon) [2][3].

Patients with coronary artery disease undergoing transluminal coronary angioplasty have been used as a model of neutrophil and platelet activation during reperfusion injury [4][5]. Neutrophil surface expression of the ß₂ integrin CD11b/CD18 (the C3bi receptor) increases after coronary angioplasty, but not after coronary arteriography [4]. Similar increases are seen with platelet CD62P (P-selectin) [5], which is a constituent of the platelet -granule and becomes expressed on the platelet surface upon activation.

Although cellular activation stimulates production of vasoconstrictive substances, the intact vascular endothelium counteracts this effect and vasodilation occurs. Alternatively, atherosclerotic coronary arteries have decreased endothelium-dependent vasodilation [6]. The most significant endothelial-based vasodilator is nitric oxide. Nitric oxide is synthesized continuously by vascular endothelium and exerts antiplatelet and antineutrophil effects [3][7].

Ischemia and reperfusion disrupt the balance between cellular activation and inhibition in the microcirculation and thus may contribute to myocardial dysfunction during cardiac operations after restoration of blood flow to a cardioplegia-arrested heart. The focus of this investigation was to measure platelet and neutrophil activation in the coronary circulation after cold cardioplegic arrest and reperfusion in cardiac surgical patients. Using flow cytometry, we analyzed coronary sinus blood samples for surface markers representative of platelet (CD62P) and neutrophil (CD11b) activation.

	Material and Methods

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Patients
With approval of the Institutional Review Board of the Brigham and Women’s Hospital, 25 patients scheduled for cardiac surgical procedures were enrolled (Table 1). No patient had evidence by history or laboratory study of a hypercoagulable or hypocoagulable state. No patient had thrombolytic therapy or coronary angioplasty within 48 hours. Three patients were disenrolled from the study because of inability to obtain or maintain access to the coronary sinus.

The extracorporeal circuit consisted of a membrane oxygenator primed with 1,500 to 2,000 mL of Plasma-Lyte and 10,000 U of porcine heparin. Heparin was administered to achieve an activated clotting time of more than 400 seconds. Cold (4°C) blood cardioplegia (whole blood with 5 to 10 mEq of potassium chloride) was given initially antegrade through the aortic root (250 mL) and then retrograde through the coronary sinus (250 mL at 20-minute intervals) at the discretion of the surgeon.

Blood Sampling
Blood samples were withdrawn from indwelling coronary sinus or radial artery catheters at three time points: (A) after heparinization but before bypass; (B) upon release of the aortic cross-clamp; and (C) 5 minutes after cross-clamp release. This final time point (C) was chosen predominantly on the basis of practicality given the need to remove the cannula expeditiously before weaning the patient from bypass. In addition, animal studies show signs of reperfusion injury (free radical production, endothelial dysfunction) to occur within seconds to minutes of reperfusion [8].

Two hundred microliters of whole blood without anticoagulant was immediately fixed in 1 mL of 1% paraformaldehyde (Sigma Chemical Co, St. Louis, MO) for 30 minutes. This sample was subsequently divided for separate platelet and neutrophil labeling and analysis.

Whole blood collected in 7.5% K₃EDTA was analyzed in duplicate for platelet and neutrophil count on an automated cell counter (Microdiff 16, Coulter Corp, Miami, FL) in the final 13 of 22 patients. Blood was not collected for a complete blood count in the initial 9 patients.

Antibodies
The monoclonal antibodies fluorescein isothiocyanate (FITC) conjugated anti-CD42b and phycoerythrin (PE) conjugated anti-CD11b were obtained from Dako Corp. (Carpenteria, CA), and PE conjugated anti-CD62P and PE conjugated mouse immunoglobulin G₁ control were obtained from Becton Dickinson (San Jose, CA).

Platelet Labeling
Platelet activation was measured by surface expression of CD62P [9]. In brief, after fixation, samples were centrifuged (2,000 g for 5 minutes at 21°C) and the pellet resuspended in phosphate-buffered saline solution (Dulbecco’s with 0.2% sodium azide; Sigma). Fifty microliters of this dilute blood suspension (1:6 vol:vol) was incubated with saturating concentrations (10 µg/mL) of FITC conjugated anti-CD42b and PE conjugated anti-CD62P for 20 minutes at room temperature in the dark. After incubation, the samples were diluted with 1 mL of phosphate-buffered saline solution, recentrifuged and the pellet resuspended in 250 µL of phosphate-buffered saline solution for analysis.

Neutrophil Labeling
After fixation and initial centrifugation as above, the blood pellet was resuspended in 100 µL of 4% fetal calf serum (Sigma) and saturating concentrations (10 µg/mL) of PE-conjugated anti-CD11b. The suspension was incubated for 20 minutes at room temperature in the dark. Erythrocytes were then lysed by vigorous vortexing in distilled water and left undisturbed for an additional 10 minutes after which the suspension was recentrifuged and the pellet resuspended in 250 µL of phosphate-buffered saline solution for flow cytometric analysis.

Flow Cytometric Analysis
Labeled cells were analyzed for PE fluorescence using a FACSort flow cytometer and CellQuest software (Becton Dickinson). Instrument calibration with Becton Dickinson Calibrite beads and FACSComp software included optimization using platelets and neutrophils labeled with isotype-matched control antibodies.

Platelets were identified in whole blood by size characteristics (forward light scatter) and by positive FITC fluorescence for anti-CD42b. Neutrophils were identified on the basis of their distinct size and cellular complexity (side scatter). Approximately 10,000 cells per sample were analyzed.

Neutrophil activation was defined in arbitrary units of PE fluorescence, whereas platelet activation was defined as the percentage of platelets expressing PE fluorescence above that of an isotype matched control.

Data Analysis
All data are expressed as mean ± standard error of the mean. Two-way analysis of variance for repeated measures was performed using Sigma Stat (Jandel Scientific, San Raphael, CA). Post hoc pairwise comparisons were made using the Tukey test with significant differences between groups defined as a p value of less than 0.05.

	Results

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The indices of activation that were measured increased during cardiopulmonary bypass (CPB), but were not further elevated 5 minutes after cross-clamp release. Specifically, the percentage of platelets expressing CD62P significantly increased by 2.8- to 3.8-fold at time points B and C in both arterial and coronary sinus blood (Fig 1). A representative platelet sample is shown in Fig 2. The increased CD62P expression on the right side of the Fig 1 corresponds to approximately 13% platelet activation. The neutrophil expression of CD11b (mean fluorescence) increased to a lesser degree, about 50% more than prebypass values, but was still significantly changed (Fig 3). A representative sample is shown in Fig 4, where the increased CD11b at the right side of the Fig 1 corresponds to a mean fluorescence intensity of approximately 97 arbitrary units of fluorescence. Platelet (but not neutrophil) activation was higher in those patients (n = 4) having combined valve and coronary artery bypass grafting procedures than those having coronary artery bypass grafting alone (p < 0.05).

View larger version (17K): [in this window] [in a new window]   Platelet activation as percent of total platelets expressing CD62P. Platelet activation increased during cardiopulmonary bypass (time points B and C). No difference was seen in coronary sinus versus arterial samples.

View larger version (18K): [in this window] [in a new window]   Representative sample of platelet CD62P expression before (left) and during (right) cardiopulmonary bypass.

View larger version (20K): [in this window] [in a new window]   Neutrophil activation by mean fluorescence for CD11b in arbitrary units of fluorescence. Neutrophil activation occurred during cardiopulmonary bypass (time points B and C), but no difference occurred between coronary sinus and arterial samples.

View larger version (16K): [in this window] [in a new window]   Representative sample of neutrophil CD11b expression before (left) and during (right) cardiopulmonary bypass.

View larger version (20K): [in this window] [in a new window]   Platelet count (x103/µL). Platelet count decreased during cardiopulmonary bypass but did not differ between coronary sinus and arterial samples.

View larger version (18K): [in this window] [in a new window]   Neutrophil count (x103/µL). Neutrophil count increased during cardiopulmonary bypass but did not differ between coronary sinus and arterial samples.

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We have demonstrated that both neutrophils and platelets become significantly activated during CPB, but do not exhibit further expression of CD11b and CD62P on transit through the coronary microvasculature after cross-clamp release. This confirms previous studies [10][11] demonstrating up-regulation of CD62P and CD11b during CPB. Separate studies in our laboratory show that platelets harvested during CPB can be further stimulated to near 100% CD62P expression by exposure to ADP in vitro (unpublished results). Clearly, platelets already activated during CPB have the capacity to increase expression of CD62P, but are not sufficiently stimulated during reperfusion to do so.

Several lines of evidence support the central role of platelet and neutrophil activation in ischemia–reperfusion injury. Platelet-activating factor, a potent platelet-activating substance, is released by the heart with ischemia and reperfusion [12]. Consistent with those findings, platelet-activating factor antagonists inhibit platelets and reduce arrhythmia and infarct size after myocardial ischemia and reperfusion [12][13]. Complement is activated with reperfusion and the anaphylatoxins C3a and C5a are generated causing neutrophil activation and expression of CD11b/CD18 [14]. This receptor interacts with the complement fragment C3bi and the endothelial ligand ICAM-1 to form a more stable neutrophil–endothelial interaction. Antibodies against the CD11b/CD18 complex, or against ICAM-1 greatly reduce the extent of reperfusion injury [15].

We were unable to demonstrate increased activation of platelets and neutrophils in coronary sinus blood after cardioplegic arrest and reperfusion. Although deposition of activated cells onto the coronary microvasculature could account for the lack of difference between sampling sites, platelet and neutrophil counts did not differ between the two sampling sites suggesting that cells were not lost on transit through the coronary circulation. In addition, although platelet activation was greater in patients having combined valve and coronary bypass procedures, this was not necessarily a result of a lengthened time of bypass. No correlation existed between cross-clamp time and platelet and neutrophil activation at any time point or sampling site.

Increased platelet and neutrophil activation resulting from CPB itself may mask activation occurring in the coronary microvasculature. It is possible that other assays of platelet and neutrophil activation, such as ß-thromboglobulin, platelet factor 4, thromboxane B₂ myeloperoxidase, lactoferrin, or elastase might have been able to detect a difference not seen with the flow cytometric assays used here. These parameters also have been shown to increase dramatically in arterial blood during CPB [16][17].

However, flow cytometric assay of surface antigens has been widely recognized as a highly sensitive indicator of both platelet and neutrophil activation [9][18]. Flow cytometry offers distinct advantages over the previously discussed assays, negating the effects of dilution seen commonly on bypass, and allowing for identification of subpopulations of activated cells within a large sample.

It is also possible that insufficient time passed to detect an effect of reperfusion in the coronary microvasculature. In some animal models, endothelial dysfunction does not occur until several hours of reperfusion have passed [19]. In contrast, angioplasty models show an almost immediate platelet and neutrophil activation after reperfusion, but in this model a traumatic (balloon), rather than reperfusion-mediated, endothelial injury may be the cause.

These data suggest that cardioplegic arrest during CPB does not induce early markers of reperfusion injury in the initial minutes after reperfusion. However, whether this model is adequate to assess the full extent of reperfusion injury associated with cardiac operations has not been determined. Previous studies differ on the possibility of a damaging effect of cardioplegia on coronary endothelium, with differences related to the potassium concentration of the cardioplegia [20][21]. The low-potassium blood cardioplegia used in this investigation has not been associated with endothelial dysfunction. In addition, study patients received predominantly retrograde cardioplegia, which may provide superior protection from ischemia [22]. Although cardioplegia may provide effective protection from ischemia during cross-clamped cardiac arrest, further study is needed to explain the cause of the reperfusion syndrome frequently seen in the postoperative period.

	Acknowledgments

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We gratefully acknowledge Jill Lamanna, BA, and Elizabeth Lawler, BA, for their technical assistance; James Gosnell, RN, and Anne Guttendorf, RN, for their help in patient recruitment; Sary Aranki, MD, for allowing the use of his patients for the study, and Gary R. Strichartz, PhD, for helpful suggestions on the manuscript.

This study was supported by the BWH Anesthesia Foundation, Inc.

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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