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ACC/AHA 2008 Performance Measures for Adults With ST-Elevation and Non-ST-Elevation Myocardial Infarction

ACC/AHA 2008 Guidelines for the Management of Adults With Congenital Heart Disease
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J Am Coll Cardiol, 1999; 33:97-106
© 1999 by the American College of Cardiology Foundation
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CLINICAL STUDIES

Chimeric 7e3 Fab (ReoPro) decreases detectable CD11b on neutrophils from patients undergoing coronary angioplasty

Judith K. Mickelson, MD, FACC* {ddagger}, M. Nadir Ali, MD* {ddagger}, Neal S. Kleiman, MD, FACC*, Nasser M. Lakkis, MD*, Thomas W. Chow, PhD§, Bonnie J. Hughes, BS{dagger} and C. Wayne Smith, MD{dagger}

* Section of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030 USA
{dagger} Speros P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine; Houston, Texas 77030 USA
{ddagger} Veterans Administration Medical Center, Houston, Texas, 77030 USA
§ Rice University, Houston, Texas, 77030, USA

Manuscript received June 3, 1998; revised manuscript received August 4, 1998, accepted September 4, 1998.

Address for correspondence: C. Wayne Smith, Room 6014, Children’s Nutrition Research Center, 1100 Bates, Houston, Texas 77030-2600


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Objectives. The purpose of this study was to monitor the effects of chimeric 7E3 Fab (ReoPro) on leukocyte and platelet activation and interaction during coronary angioplasty.

Background. Increased expression of CD11b on monocytes and neutrophils promotes their adhesion to endothelial cells, extracellular matrix and smooth muscle cells. Thrombin-activated platelets adhere via P-selectin to monocytes and neutrophils. These cell interactions may affect the outcome of coronary angioplasty.

Methods. During coronary angioplasty, venous blood was obtained for flow cytometric detection of leukocyte CD11b; platelet CD41a, CD61a and CD62P; the percentage of leukocytes with adherent platelets and the intensity of bound platelet fluorescence.

Results. Leukocyte CD11b expression increased after angioplasty in control patients (neutrophils 171 ± 25 to 255 ± 31 mean fluorescence intensity [MFI, mean ± SEM], n = 25, p < 0.0001; monocytes 200 ± 40 to 248 ± 36 MFI, n = 17, p < 0.05) and decreased in the patients selected to receive chimeric 7E3 Fab (neutrophils 146 ± 30 to 82 ± 22 MFI, n = 25, p < 0.0001; monocytes 256 ± 53 to 160 ± 38 MFI, n = 17, p < 0.05). Neutrophil CD11b decreased after in vitro incubation of whole blood with chimeric 7E3 Fab (n = 5, p = 0.01), but fMLP-induced increases in CD11b were not prevented. The CD11b expression was unchanged and increased with fMLP stimulation after in vitro incubation of isolated neutrophils with chimeric 7E3 Fab. Direct-labeled chimeric 7E3 Fab was not detected bound to neutrophils in whole blood or isolated cells using flow cytometric techniques. Adhesion of isolated neutrophils to protein-coated glass was not prevented by in vitro incubation with chimeric 7E3 Fab. Platelet activation increased after angioplasty in control patients (CD62P 8.9 ± 0.8 to 12.3 ± 1.2 MFI, n = 25, p < 0.05; CD41a 382 ± 25 to 454 ± 26 MFI, n = 25, p < 0.05, CD61a 436 ± 52 to 529 ± 58 MFI, n = 11, p < 0.05); it did not increase in the patients selected to receive chimeric 7E3 Fab (CD62P 13.2 ± 1.0 to 9.0 ± 0.9 MFI, n = 25, p < 0.05; CD61a 398 ± 32 to 410 ± 38 MFI, n = 7, p = NS). Leukocytes with adherent platelets tended to increase in the control group of patients and decrease after the procedure in patients selected to receive chimeric 7E3 Fab; individual and procedure-related variability were marked.

Conclusions. Despite standard aspirin and heparin therapy, leukocyte and platelet activation with platelet adherence to leukocytes occurs after coronary angioplasty. Although chimeric 7E3 Fab does not bind to leukocytes directly, it influences CD11b expression in whole blood. Modulation of platelet and leukocyte activation and interaction by chimeric 7E3 Fab may contribute to an improved outcome after coronary angioplasty.

Coronary angioplasty is a routinely used invasive modality for the treatment of stable angina, unstable angina and acute myocardial infarction. While the initial success rate is very high, long-term and event-free survival has been limited by restenosis and progression of atherosclerotic disease (1,2). New devices, in particular the heparin-coated stent with ticlopidine, have shown improved event-free survival in patients with stable angina (3,4). Recent studies with chimeric 7E3 Fab (antibody directed against a ß3integrin [{alpha}IIbß3, glycoprotein IIb/IIIa]) have shown very encouraging clinical results early and late after both high- and low-risk coronary angioplasty (5,6).

Platelet adhesion and aggregation play significant roles in acute coronary syndromes (7–9). Fibrinogen, fibronectin, vitronectin, or von Willebrand Factor binding to platelet membrane glycoprotein (GP) IIb/IIIa initiate platelet functional responses (10,11). Thrombin-activated platelets adhere to leukocytes via P-selectin (12–14). The interaction of platelets with leukocytes involves P-selectin glycoprotein ligand-1 (PSGL-1) and possibly CD11b/CD18 (Mac-1), which interacts with fibrinogen bound to platelets via GP IIb/IIIa or possibly tethering on platelet ICAM-2 (15–17). Markers of thrombin generation and activity have been reported in patients undergoing coronary angioplasty (17). The detection of activated platelets in clinical disorders, including unstable coronary syndromes or coronary interventional procedures, has been described (18–21). Several studies have found leukocyte and platelet activation under similar circumstances (22–28). The dynamics of leukocyte–platelet interactions have been studied during cardiac procedures (cardiopulmonary bypass or coronary angioplasty) or in patients with coronary artery disease (16,26,29–32). Leukocyte activation or leukocyte-platelet complexes may participate in the vascular inflammatory process, including restenosis, following coronary angioplasty (25,32,33).

The CD11b/CD18 (Mac-1) is a heterodimer in the ß2 integrin family ({alpha}Mß2) that promotes adhesion of neutrophils and monocytes to intercellular adhesion molecule 1 (ICAM-1) on endothelial cells, extracellular matrix, iC3b and components of the coagulation cascade (e.g., Factor X and fibrinogen). It is stored in secretory granules in these leukocytes and can be rapidly mobilized to the cell surface following cell activation. Constitutive surface levels of CD11b are relatively low, with approximately 50,000 binding sites per cell (34). Neutrophil activation may increase surface levels of Mac-1 four- to fivefold. Flow cytometric determination of leukocyte surface levels of CD11b has been used as an index of activation of these cells. The purpose of this study was to determine whether peripheral blood leukocyte and platelet activation and interaction are affected by administration of chimeric 7E3 Fab (ReoPro) during coronary angioplasty.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Patient population.   The study group consisted of 50 patients undergoing elective coronary angioplasty. All of the patients were stable at the time of angioplasty, having presented to the hospital with progressive angina, unstable angina or recent acute myocardial infarction. All medications (aspirin, heparin, nitrates, calcium channel or beta-blockers) were continued throughout the hospitalization. Baseline blood studies obtained prior to the procedure included CBC, differential, platelet count, PT/PTT, chemistry profiles, and lipoprotein profile. This study was approved by the Institutional Review Boards of Baylor College of Medicine and the Houston Veterans Administration Medical Center, and all patients gave written informed consent.

Angioplasty protocol.   After local anesthesia (2% lidocaine, 20 cc), a 6F sheath was placed in the femoral vein and an 8F sheath was placed in the femoral artery. The use of chimeric 7E3 Fab (ReoPro, Centocor, Leiden, The Netherlands) during coronary angioplasty was based on the clinical judgment of the individual operator and followed the manufacturer’s recommendations (0.25 mg/kg intravenous bolus and 10 µg/min infusion for 12 h), including heparin dose adjustments. After discarding the initial 3-cc aspirate, blood was collected into sterile plastic syringes from the venous sheath and placed in vacutainers with 3.2% buffered citrate solution. The preprocedural sample was obtained before administration of heparin, chimeric 7E3 Fab, or contrast material, and the postprocedural sample was obtained after administration of all materials (35). In six patients given chimeric 7E3 Fab, venous blood samples were obtained 12 and 24 h after the procedure.

Monoclonal antibodies.   With the exception of the anti-CD18 monoclonal antibody (R15.7 provided by Dr. R. Rothlein, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut), the monoclonal antibodies were commercially available, purified whole immunoglobulins. All studies included isotype-specific irrelevant monoclonal antibodies with the appropriate fluorescent label as controls (Becton Dickinson Immunocytometry Systems, San Jose, California). The following antibodies were used for platelet studies: anti-CD41a fluorescein isothiocyanate conjugated (FITC), which recognizes IIb in the intact complex with IIIa of platelet GP IIb/IIIa but not with IIb or IIIa separately (AMAC, Westbrook, Maine); anti-CD61a fluorescein isothiocyanate conjugated (FITC), which recognizes IIIa of platelet GP IIb/IIIa (DAKOpatts, Denmark); and anti-CD62P phycoerythrin conjugated (PE), which recognizes the 140-kD single-chain GMP-140 (P-selectin) of platelet membrane granule glycoprotein on activated platelets (Serotec Ltd, Oxford, England). The following antibodies were used for platelet–leukocyte studies: anti-CD61a FITC or anti-CD41a FITC with either anti-CD45RO phycoerythrin conjugated (PE), which recognizes a CD45 isoform present on neutrophils, monocytes, and lymphocytes but neither erythrocytes nor platelets (DAKOpatts, Denmark), or anti-CD14 phycoerythrin conjugated (PE), which recognizes Mo2 present on monocytes (Becton Dickinson Immunocytometry Systems, San Jose, California). Neutrophil CD11b expression was studied with two phycoerythrin-conjugated monoclonal antibodies (Leu-15, Becton Dickinson Immunocytometry Systems; 2LPM19c, DAKOpatts, Denmark).

Neutrophil studies.   Venous blood samples were collected from healthy adult individuals taking no medications to assess the possible interaction of the chimeric 7E3 Fab with CD11b/CD18 (Mac-1) on neutrophils. Each blood sample was divided and anticoagulated with either heparin or citrate. Subsequent comparisons showed no difference between the two anticoagulants in individual neutrophil CD11b expression with the two phycoerythrin-conjugated monoclonal antibodies studied (Leu-15, Becton Dickinson Immunocytometry Systems; 2LPM19c, DAKOpatts, Denmark). Chimeric 7E3 Fab was added to whole blood or isolated neutrophils at a final concentration of 3.5 µg/ml and incubated at 37°C for 30 min (optimal inhibitory concentration of neutrophil CD11b expression based upon studies ranging from 0.35 µg/ml to 350 µg/ml). Isolated neutrophils were prepared from citrate anticoagulated, dextran-sedimented venous blood over Ficoll-Hypaque gradients and were suspended in Dulbecco’s phosphate-buffered saline (PBS, Gibco, Grand Island, New York), pH 7.4, containing 0.2% dextrose as previously described (36).

The isolation steps were carried out at room temperature, and then neutrophils were maintained at room temperature in PBS at a concentration of 107/ml. Isolated neutrophil preparations were confirmed to be free of platelet contamination (anti-CD41a FITC) using flow cytometry (mean fluorescence intensity similar to background and anti-IgG control) and direct visualization of fluorescent platelets attached to neutrophils (<1% neutrophils with any [≤2] platelets). For in vitro activation studies, neutrophils were stimulated with 10 nM f-Met-Leu-Phe (fMLP). Isolated neutrophils were incubated with chimeric 7E3 Fab (3.5 µg/ml) for 0, 5, 15, 30, 45 or 60 min at 37°C prior to incubation with monoclonal antibody (2LPM19c PE) for 30 min at 4°C. There was no change in fluorescence intensity over this time course of incubation, suggesting that chimeric 7E3 Fab did not cause internalization or shedding of CD11b from the isolated neutrophils. There was no difference in fluorescence intensity of isolated neutrophils stained for CD11b expression (2LPM19c PE) when studies were carried out at 4°C (15 or 30 min) with PBS compared to chimeric 7E3 Fab.

Neutrophil adhesion assays.   Round coverglasses (25 mm) were coated with keyhole limpet hemocyanin (KLH; Sigma Chemica, St. Louis, Missouri) and mounted in adhesion chambers as previously described (37,38). Isolated neutrophils suspended in PBS were allowed to settle onto this surface for 500 s, and then the chamber was inverted for an additional 500 s. The percentage of neutrophils remaining attached was determined. Adhesion of isolated neutrophils without stimulation was compared to fMLP-stimulated cells after incubation (37°C for 15 min) with chimeric 7E3 Fab (3.5 µg/ml), anti-CD18 (R15.7), or anti-IGg1 control.

Direct-labeled chimeric 7E3 Fab studies.   To determine whether there was direct binding of the chimeric 7E3 Fab to neutrophils, either in whole blood or isolated preparations, citrate anticoagulated venous blood samples were collected from healthy adult individuals taking no medications. Chimeric 7E3 Fab (Lot 96D03AA, ReoPro, Centocor, Leiden, The Netherlands) was conjugated with fluorescein isothiocyanate using a commercially available kit (FITC Conjugation Kit, Boehringer Mannheim Corp, Indianapolis, Indiana). Fluorescence saturation was determined by titration of the chimeric 7E3 Fab-labeled preparation with isolated platelets (5 x 107/ml) stimulated with ADP (20 µmol/liter). Live flow cytometric analysis of whole blood or isolated neutrophil preparations was performed using intravital nuclear staining (LDS 751, Molecular Probes, Eugene, Oregon) to allow triggering on neutrophils. Chimeric 7E3 Fab FITC and Leu-15 PE binding to neutrophils in whole blood or isolated preparations was quantitated for both stimulated (10 nmol/liter fMLP) and unstimulated neutrophils. The controls were anti-IGg1 FITC and anti-IGg2a PE.

To determine whether there was direct binding of the chimeric 7E3 Fab to monocytes, venous whole-blood samples anticoagulated with EDTA were collected from healthy adult individuals taking no medications. Next, 100 µl of anticoagulated whole blood was incubated with saturating concentrations of the antibody (chimeric 7E3 Fab FITC or anti-IGg1 FITC control). After a 20-min incubation in the dark at room temperature, cells were washed (Tyrode’s-HEPES buffer), resuspended in 1 ml of lysing reagent (Becton Dickinson Immunocytometry Systems), mixed, incubated in the dark for 10 min at room temperature, and washed (TH-BSA). Cells were resuspended in 200µl of 1% paraformaldehyde and analyzed immediately. Measurement of monocyte FITC fluorescence was done by live-gating on leukocyte-sized events, using forward- versus side-scatter parameters to distinguish monocytes.

Fluorescence labeling.   For platelet or platelet–leukocyte studies, the anticoagulated whole blood was immediately placed in an equal volume of 2.5% paraformaldehyde at room temperature for 5 min and then incubated at 4°C. After 1 h, Tris-glycine solution, 1/8:v/v (250 mmol/liter Tris and 500 mmol/liter glycine) was added (to stop fixation). Samples were kept refrigerated until the day’s study was completed (<3 h). Samples were washed in Tyrode’s-HEPES buffer (HEPES 5 mmol/liter, NaCl 140 mmol/liter, KCl 2.7 mmol/liter, dextrose 5.5 mmol/liter, NaH2PO4 0.42 mmol/liter and NaHCO3 12 mmol/liter, pH 7.4) with 2 mg/ml of bovine serum albumin (TH-BSA) and resuspended to the original volume. For leukocyte and platelet activation studies, the anticoagulated whole blood was refrigerated immediately without fixation.

For platelet studies, 5 µl of the fixed washed whole blood was added to 40 µl of Tyrode’s-HEPES buffer and incubated with saturating concentrations of one of the antibodies: anti-CD41a FITC, anti-CD61a FITC, anti-CD62P PE, or anti-IGg1 (FITC and PE) controls. For platelet-activation studies, 5 µl of anticoagulated unfixed whole blood was added to 35 µl of Tyrode’s-HEPES buffer with 5 µl of either ADP (10 µmol/liter final concentration) or thrombin receptor activating peptide (7.5 µmol/liter final concentration [TRAP]) and incubated with saturating concentrations of the antibodies as described above; TRAP, the 11-amino acid (SFLLRNPNDKY-NH2) thrombin receptor-activating peptide, was obtained from Peninsula Laboratories Europe Limited (Merseyside, England). After a 20-min incubation in the dark at room temperature, cells were resuspended in 2 ml of 1% paraformaldehyde and stored in the dark at 4°C in 5 ml polystyrene snap cap tubes until they could be read (<3 days). For leukocyte–platelet studies, 100 µl of the fixed washed whole blood was incubated with saturating concentrations of the antibodies: anti-CD61a or anti-CD41a FITC and anti-CD45R0 PE, anti-IGg1 FITC control and anti-CD45R0 PE, anti-CD61a or anti-CD41a FITC and anti-CD14 PE, or anti-IGg1 FITC control and anti-CD14 PE. For the neutrophil CD11b expression studies, 100 µl of anticoagulated unfixed whole blood or isolated neutrophils was incubated with saturating concentrations of the antibody (Leu 15 PE, 2LPM19c PE, or anti-IGg2a PE control). After a 20-min incubation in the dark at room temperature, cells were washed (Tyrode’s-HEPES buffer). The whole blood or washed whole-blood samples were resuspended in 1 ml of lysing reagent (Becton Dickinson Immunocytometry Systems), mixed, incubated in the dark for 10 min at room temperature, and washed (TH-BSA). For leukocyte–platelet studies or neutrophil studies, cells (from lyzed whole blood or isolated preparations) were resuspended in 200 µl of 1% paraformaldehyde and stored in the dark at 4°C in 5 ml polypropylene snap cap tubes until they could be read (<3 days).

Flow cytometry.   Samples were analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Platelet surface CD41a, CD61a and CD62P (GP IIb/IIIa and P-selectin, respectively) were measured by live gating on platelet-sized events, using forward- versus side-scatter parameters in log scale amplification to distinguish platelets from erythrocytes. Three thousand platelet events were collected and evaluated. For leukocyte–platelet studies, live gating was performed on leukocyte-sized events (either neutrophils based on the anti-CD45R0 PE-labeled population or monocytes based on the anti-CD14 PE-labeled population). Assessment of 10,000 events for neutrophils and 5,000 events for monocytes was performed. The percentage of leukocytes with positive platelet fluorescence (FITC) and the mean platelet marker fluorescence intensity of the leukocytes were recorded. Measurements of leukocyte-surface CD11b was done by live gating on leukocyte-sized events, using forward- versus side-scatter parameters to distinguish neutrophils or monocytes. Ten thousand events for neutrophils and 5,000 events for monocytes were evaluated.

Statistical analysis.   Data are presented as mean ± SEM. Comparisons between angioplasty study groups and sampling times were made by analysis of variance. The Newman-Keuls multiple comparison test was used to determine which means were significantly different. Paired t test was used to compare before and after angioplasty results for individual platients. Time-dependent changes were determined by two-way repeated measures ANOVA. If the time factor main effect was significant, a Newman-Keuls multiple comparison test was used to determine which means were significantly different. Contingency tables and chi-square with continuity correction were used to compare study groups with respect to sex, race, clinical circumstances, involved artery, risk factors, procedure performed, and restenosis. A p ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Patient population.   There were 50 patients studied (age 58 ± 1 yr), 25 undergoing coronary angioplasty with and 25 without administration of chimeric 7E3 Fab (Table 1). Patients selected to receive chimeric 7E3 Fab were younger. There were no significant differences between the two treatment groups with respect to sex or race. Racial composition of the group included 12 blacks, 5 Hispanics and 33 whites. There was no difference in age between the sexes or the racial groups. White blood cell counts (8.8 ± 0.5 x 103/mm3), differentials (neutrophils 68 ± 2%, lymphocytes 24 ± 2%, monocytes 6 ± 1%) and platelet counts (234 ± 10 x 103/mm3) were within normal ranges and not different between treatment groups. Hypertension (78%), smoking (30%) and diabetes (36%) were common in both groups. Chimeric 7E3 Fab tended to be administered more often to patients with right coronary artery procedures (Table 2). Heparin dose was expectedly lower with administration of chimeric 7E3 Fab.


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  		Table 1 Baseline Patient Characteristics

 

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  		Table 2 Procedure Characteristics

 
Leukocyte studies.   Leukocyte CD11b expression was similar in both study groups prior to the procedure. The CD11b expression increased after angioplasty in control patients (neutrophils 171 ± 25 to 255 ± 31 mean fluorescence intensity [MFI, mean ± SEM], n = 25, p < 0.0001; monocytes 200 ± 40 to 248 ± 36 MFI, n = 17, p < 0.05) and decreased in the patients selected to receive chimeric 7E3 Fab (neutrophils 146 ± 30 to 82 ± 22 MFI, n = 25, p < 0.0001; monocytes 256 ± 53 to 160 ± 38 MFI, n = 17, p < 0.05 [Fig. 1]). After the procedure with chimeric 7E3 Fab, leukocyte CD11b expression was lower than the control group.



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  		Figure 1 Leukocyte CD11b expression. Peripheral blood samples obtained pre- and postangioplasty were compared (paired t test). (a) Neutrophil CD11b expression in the control group increased (n = 25, p < 0.0001), while neutrophil CD11b expression decreased in the chimeric 7E3 group (n = 25, p < 0.0001). Postprocedure, neutrophil CD11b expression was lower in the chimeric 7E3 Fab group than the control group (p < 0.0001). (b) Monocyte CD11b expression in the control group increased (n = 17, p < 0.05), while monocyte CD11b expression decreased in the chimeric 7E3 group (n = 17, p < 0.05). Postprocedure, monocyte CD11b expression was lower in the chimeric 7E3 Fab group than in the control group (p < 0.05).

 
To assess further the possible interaction of the chimeric 7E3 Fab with CD11b/CD18 (Mac-1) on neutrophils, a number of in vitro studies were performed using venous blood samples from healthy adult individuals taking no medications. In fact, when chimeric 7E3 Fab was incubated with the whole blood at 37°C, there was a significant decrease in neutrophil CD11b detected in paired samples by flow cytometric techniques with two different phycoerythrin-conjugated monoclonal antibodies (p = 0.01, Fig. 2). However, the expected increase in neutrophil CD11b expression after fMLP stimulation (10 nmol/liter) was not prevented by incubation of chimeric 7E3 Fab with the whole blood at 37°C (Fig. 2). This suggests that the chimeric 7E3 Fab may not be acting directly on the CD11b receptor. Isolated neutrophils, free of platelet contamination, were studied so as to define this interaction further. There was no difference in the baseline or fMLP-stimulated CD11b expression on isolated neutrophil in vitro with or without chimeric 7E3 Fab (Fig. 3).



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  		Figure 2 Neutrophil CD11b expression. Peripheral blood samples obtained from healthy adult volunteers, incubated with 3.5 µg/ml chimeric 7E3 Fab at 37°C for 30 min, showed a decrease in each individual’s neutrophil CD11b expression (paired t test, n = 5). Peripheral whole blood samples obtained from healthy adult volunteers were incubated with 3.5 µg/ml chimeric 7E3 Fab at 37°C for 30 min and then stimulated with fMLP (10 nmol/liter) to determine if this apparent decrease in detectable CD11b was of functional significance. After stimulation, neutrophil CD11b expression increased dramatically regardless of the presence of chimeric 7E3 Fab (paired t test, n = 5). Two phycoerythrin-conjugated monoclonal antibodies were studied to confirm this finding (Leu-15, Becton Dickinson Immunocytometry Systems, San Jose, California; 2LPM19c, DAKOpatts, Denmark).

 


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  		Figure 3 Isolated neutrophil CD11b expression. Neutrophils isolated from peripheral blood of healthy adult volunteers were incubated with 3.5 µg/ml chimeric 7E3 Fab at 37°C for 30 min and then stimulated with fMLP (10 nmol/liter). There was no difference in CD11b expression with or without chimeric 7E3, either before or after stimulation with fMLP (paired t test, n = 5). Two phycoerythrin-conjugated monoclonal antibodies were studied to confirm this finding (Leu-15, Becton Dickinson Immunocytometry Systems, San Jose, California; 2LPM19c, DAKOpatts, Denmark).

 
Adhesion of neutrophils is mediated by the ß2integrins, in particular CD11b/CD18. Under certain conditions, CD11a/CD18 or CD11c/CD18 may be involved (39). Adhesion to protein-coated surfaces is a reasonable surrogate used to assess the CD11b functional properties and the impact of specific monoclonal antibodies on them. Isolated neutrophils from healthy adult individuals taking no medications (n = 4) were used for the adhesion assay. Adhesion of neutrophils in the presence of a control monoclonal antibody was very low (5.2 ± 1.9%) and increased significantly after fMLP stimulation (51.8 ± 6.9%, p < 0.05). The adhesion of stimulated neutrophils was prevented by anti-CD18 (7.3 ± 3.0%), which blocks all ß2integrins, but not by chimeric 7E3 Fab (56.7 ± 5.9%).

Finally, it seemed most important to determine whether a difference existed between neutrophils in whole blood or in isolated preparations with respect to possible binding of chimeric 7E3 Fab to the CD11b receptor. The FITC-labeled chimeric 7E3 Fab was not detected bound to neutrophils using live flow cytometric analysis of whole blood or isolated preparations, with or without fMLP stimulation. Furthermore, the FITC-labeled chimeric 7E3 Fab was not detected bound to monocytes using live flow cytometric analysis of EDTA-anticoagulated whole blood.

Platelet studies.   Platelet activation increased after angioplasty in control patients (CD62P 8.9 ± 0.8 to 12.3 ± 1.2 MFI, n = 25, p < 0.05; CD41a 382 ± 25 to 454 ± 26 MFI, n = 25, p < 0.05, CD61a 436 ± 52 to 529 ± 58 MFI, n = 11, p < 0.05) and decreased in the patients selected to receive chimeric 7E3 Fab (CD62P 13.2 ± 1.0 to 9.0 ± 0.9 MFI, n = 25, p < 0.05; CD41a 365 ± 29 to 36 ± 7 MFI, n = 25, p < 0.0001, CD61a 398 ± 32 to 410 ± 38 MFI, n = 7, p = NS [Fig. 4]). Platelet CD62P expression was higher before and lower after the procedure in patients selected to receive chimeric 7E3 Fab compared to the control group (p < 0.01). In both groups, in vitro stimulation with TRAP or ADP, either before or after the angioplasty, caused significant platelet activation, with an increase in the three measured surface receptors (all p < 0.0001, Table 3). Detection of in vitro-stimulated increase in GP IIb/IIIa was limited when chimeric 7E3 Fab was administered in vivo and anti-CD41a was used for detection (p < 0.0001).



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  		Figure 4 Platelet studies. Peripheral blood samples obtained pre- and postangioplasty were compared (paired t test). (a) Platelet P-selectin (CD62) expression in the control group increased (n = 25, p < 0.05), while platelet P-selectin (CD62) expression decreased in the chimeric 7E3 group (n = 25, p < 0.05). Preprocedure platelet P-selectin (CD62) expression was higher and postprocedure platelet P-selectin (CD62) expression was lower in the chimeric 7E3 Fab group than the control group (p < 0.01). (b) Platelet GP IIb/IIIa expression (as detected by anti-CD41a) in the control group increased (n = 25, p < 0.05), while the GP IIb/IIIa receptor was not accessible to the anti-CD41a in the chimeric 7E3 group (n = 25, p < 0.0001).

 

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  		Table 3 Ex vivo Platelet Stimulation Studies*

 
Time course of chimeric 7e3 Fab.   To assess the duration of the chimeric 7E3 Fab’s biologic influence on platelets and leukocytes, six patients were studied at 12 h (the end of the infusion) and 24 h. These patients were clinically stable and asymptomatic during this time period. The decrease in leukocyte CD11b expression persisted, as did the decline in platelet activation (Table 4). The GP IIb/IIIa receptor remained inaccessible to the anti-CD41a throughout this period.


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  		Table 4 Time Course of Chimeric 7E3 Fab Effect on Detectable Platelet and Leukocyte Receptors

 
Leukocyte–platelet interactions.   Cytometric histograms showed three peaks (arbitrarily called low, high, and very high) of platelet fluorescence intensity on both monocytes and neutrophils (32). The percent of neutrophils with more adherent platelets tended to be higher in the control group both before and after the procedure (Table 5). Both the mean platelet fluorescence intensity of the leukocytes (i.e., the number of platelets per leukocyte) and the percentage of leukocytes with positive platelet fluorescence fell to near zero after chimeric 7E3 Fab because it blocked access of the antibody used to detect the platelets (GP IIb/IIIa receptor [anti-CD41a]). When anti-CD61a was used to detect the platelets, the increase in leukocytes with more adherent platelets after the procedure still occurred in the control group but not with chimeric 7E3 Fab present.


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  		Table 5 Leukocyte–Platelet Interactions*

 
Subsequent studies.   Nineteen patients (7 chimeric 7E3 Fab and 12 control, p = 0.11) returned to the hospital with chest pain and underwent repeat cardiac catheterization at a median follow-up time of 16 months (range 3 to 26 months). Findings in these cases were as follows: 7 had mild restenosis (<50%) with progression of disease elsewhere and were treated medically; 4 had significant restenosis and underwent repeat angioplasty; 5 had significant new stenosis and underwent angioplasty; and 3 were referred for coronary artery bypass grafting because of significant restenosis and progression of disease elsewhere. The incidence of significant restenosis was lower in the chimeric 7E3 group (1 chimeric 7E3 Fab and 6 control, p < 0.01). There was one death (2%) during this time. This patient had diabetes and an ischemic cardiomyopathy (EF 25%). He developed restenosis 9 weeks after chimeric 7E3 Fab infusion during rotoblator and stent placement in an ostial saphenous vein graft to the obtuse marginal artery. During the cardiac catheterization defining the restenosis, the patient developed ventricular fibrillation and could not be resuscitated.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The long-term success of coronary angioplasty has been limited by restenosis (40). Various pharmacologic strategies have not eliminated this problem (41). One promising pharmacologic therapy for the reduction in clinical restenosis has been the use of chimeric 7E3 Fab (5,6). Although the most obvious mechanism by which this drug reduces the incidence of restenosis involves platelet inhibition, the biologic impact may be much greater owing to cross specificity with other integrins. Aspects of the restenotic process that could be influenced include inflammatory cells, vascular endothelium, smooth muscle cell proliferation, and the coagulation cascade (42–44).

Leukocyte studies.   Results from this study suggest, as other investigators have found, that leukocyte activation (i.e., increased CD11b expression) can be detected in peripheral blood after routine coronary angioplasty when chimeric 7E3 Fab is not used (23–25,27,28). It is unlikely that the activation state would diminish during angioplasty. However, when chimeric 7E3 Fab was administered, leukocyte CD11b expression, especially on neutrophils, diminished and remained low for some time. A prolonged interaction of the chimeric 7E3 Fab with platelets from normal subjects has already been reported (45).

This diminution in CD11b expression on neutrophils occurs in vivo or in vitro when whole blood is incubated at body temperature with the chimeric 7E3 Fab. The CD11b expression of isolated neutrophils incubated at 37°C with chimeric 7E3 Fab did not diminish. No change occurred in CD11b expression on isolated neutrophils over time, suggesting that chimeric 7E3 Fab did not cause internalization or shedding of CD11b from the isolated neutrophils. The 3.5 µg/ml concentration used (based on in vitro titration of the effect of chimeric 7E3 Fab on neutrophil CD11b expression in whole blood from healthy adult individuals taking no medications) was similar to the 2.4 ± 0.4 µg/ml detected 5 min after the standard bolus dose in patient undergoing elective coronary angioplasty (46).

Changes in CD11b expression, either on isolated neutrophils or neutrophils or monocytes in whole blood, did not appear to involve direct interactions of the FITC-labeled chimeric 7E3 Fab with the receptor. This is in contrast to in vitro studies, in which the whole murine monoclonal antibody (7E3) appeared to attach to CD11b of ADP-stimulated peripheral blood monocytes or a cultured monocytic cell line (47). Monocytes have a much higher affinity for platelet binding than do neutrophils (14,29). Thus, the possibility that these monocytes were contaminated with platelets, which are clearly activated by ADP and would themselves bind the antibody, must be excluded. Our attempts to isolate monocytes that were not activated or contaminated with platelets were not successful. Thus, we can only report on the monocytes and neutrophils in whole blood, or neutrophils that were isolated with minimal activation or platelet contamination.

A recent investigation reported by Simon et al. (48), studied the interaction of the 7E3 antibody and monocytic cells (two different cell lines [erythroleukemic K562 cells and CHO cells] transfected with the {alpha}M subunit of Mac-1 [CD11b]). The 7E3 antibody was detected bound directly to the K562 cells using flow cytometric techniques. Both the 7E3 and chimeric 7E3 Fab blocked two Mac-1 (CD11b)-dependent adhesive properties (adhesion to fibrinogen of a monocytic cell line [THP-1 cells] and adhesion to ICAM-1 of transfected CHO cells). Our inability to document antibody binding to monocytes or neutrophils may be related to differences in the two antibodies (whole murine vs. chimeric Fab) or differences in the cell types (neutrophils vs. monocytes versus transfected cell lines) with respect to Mac-1 (CD11b) binding. Although we did not find any functional changes in isolated neutrophils (ex vivo adhesion assay) or fMLP responsiveness of either isolated neutrophils or neutrophils in whole blood induced by chimeric 7E3 Fab, this does not exclude an effect on neutrophil functional responses in vivo.

Platelet studies.   Changes in platelet P-selectin (CD62P) were small but directional; thus, paired analysis showed significant difference between the patient groups: increases after routine angioplasty and decreases when chimeric 7E3 Fab was used. Several investigators have found platelet P-selectin to be increased after coronary angioplasty (49,50). After routine balloon angioplasty for stable angina, Serrano et al. (28) found evidence of increased platelet GP IIb/IIIa expression, which is another marker of platelet activation (51). In our study, increases in the platelet GP IIb/IIIa receptor density was documented with two different monoclonal antibodies (anti-CD41a recognizes IIb in the intact complex with IIIa of platelet GP IIb/IIIa but not with IIb or IIIa separately; anti-CD61a recognizes IIIa of platelet GP IIb/IIIa). After chimeric 7E3 Fab and the angioplasty procedure, surface expression of platelet P-selectin declined and platelet GP IIb/IIIa did not increase (as detected by anti-CD61a). Chimeric 7E3 Fab does not react directly with platelet surface P-selectin or the site of anti-CD61a binding; however, in vivo it may ameliorate some of the platelets’ responsiveness to agonists that do increase either receptor’s surface expression. While unlikely, perhaps more activated platelets were removed from the peripheral circulation or more surface P-selectin was lost when chimeric 7E3 was used and thus the lower level of platelet P-selectin.

In both groups, in vitro platelet stimulation with TRAP or ADP, either before or after the angioplasty, caused a significant increase in platelet P-selectin. This finding suggests that in vitro the chimeric 7E3 Fab had no direct effect on the internal pool of P-selectin or its mobilization to the cell surface. Platelet GP IIb/IIIa, as detected by anti-CD41a, was effectively blocked for 24 h by the bolus followed by 12-h infusion of chimeric 7E3 Fab, which has been described (45). Kleiman et al. (46) found inhibition of ex vivo platelet aggregation (TRAP, ADP, collagen) early after only the bolus of chimeric 7E3 Fab, but aggregation returned to 70% of baseline by 24 h. The concentration of chimeric 7E3 Fab present in the whole blood from our patients, which was diluted 1:10 for the platelet flow cytometry studies, was adequate to block detection (anti-CD41a) of many of the newly externalized GP IIb/IIIa receptors stimulated with TRAP or ADP. The flow cytometric detection (anti-CD61a) of newly externalized GP IIb/IIIa receptors, after stimulation with TRAP or ADP, was similar in both treatment groups. Thus, the chimeric 7E3 Fab did not prevent platelet activation in vitro with these two agonists.

Leukocyte–platelet interactions.   Involvement of leukocytes in coronary atherosclerosis and thrombosis has been demonstrated in several recent clinicopathologic studies (44,52). Adhesion molecules of the immunoglobulin superfamily, intercellular adhesion molecule-1 (ICAM-1 [CD54]) and vascular cell adhesion molecule-1 (VCAM-1 [CD106]), are expressed by intimal neovasculature of atherosclerotic plaques and associated with an increased intimal leukocyte accumulation probably via CD11b (Mac-1). Platelets and locally derived thrombin have been associated with acute coronary syndromes, nonocclusive thrombus formation during angioplasty, and restenosis. Patients with active coronary artery disease have enhanced in vivo adhesive interactions between platelets and leukocytes (14,26,32,53). The complexes may modulate the proinflammatory and prothrombotic effects of neutrophils and monocytes. In this study, patients selected to receive chimeric 7E3 Fab had evidence of fewer leukocyte–platelet complexes than did the control group after the angioplasty. This is not surprising as both leukocyte CD11b and platelet P-selectin expression also diminished, suggesting that passivation of both cell types may have occurred.

Study limitations.   The most serious limitation of this study is the small number of patients studied in a nonrandomized fashion. Another major limitation is the incompleteness of some data. Seventeen patients in each treatment group had an adequate number of monocytes in the peripheral blood to obtain good flow cytometric data. Studies with two GP IIb/IIIa antibodies were not obtained on all patients, but enough studies were performed to allow analysis of the data. A third issue involves the technical aspects of flow cytometric analysis of peripheral blood. It is virtually certain that what is happening in the coronary artery at and distal to the lesion is different from what we are able to study in the peripheral blood. Technically sampling blood at the site of angioplasty and distal to it is difficult and would add delays and possibly risks to the procedure. Sampling from the coronary vein, after the blood has passed through the capillary bed and venous circulation, may not be much different from peripheral blood, and again this would add delays and possibly risks to the procedure. Variations occur, and comparing absolute fluorescence intensities, as we have done, is problematic. However, each antibody was consistently obtained from a single manufacturer and carefully titrated. There was little change in absolute fluorescence intensity of CD11b, CD41a, CD61a and CD62P expression of the control subjects routinely used for clinical determinations during this time.

Conclusions.   Despite standard aspirin and heparin therapy, leukocyte and platelet activation with platelet adherence to leukocytes occurs after coronary angioplasty. Although chimeric 7E3 Fab does not bind to leukocytes directly, it influences CD11b expression on leukocytes in whole blood. In this small nonrandomized study, the incidence of significant restenosis was lower in the chimeric 7E3 group. Thus, modulation of platelet and leukocyte activation and interaction by chimeric 7E3 Fab may contribute to an improved outcome after coronary angioplasty.


   Footnotes
 
This work was supported in part by grants from the National Institutes of Health AI19031 and HL42550 (CWS).


   References
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 Abstract
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 References
 

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