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Ann Thorac Surg 2005;80:251-257
© 2005 The Society of Thoracic Surgeons

Original article: Cardiovascular

Platelet Preservation With a Glycoprotein IIb/IIIa Inhibitor in a Porcine Cardiopulmonary Bypass Model

Norihiro Kondo, MDa,*, Yasuyuki Suzuki, MDa, Fuminori Wakayama, MDa, Yoshiko Tamai, MDb, Kaiqiang Ji, MDa, Kozo Fukui, MDa, Ikuo Fukuda, MDa

a First Department of Surgery, Hirosaki University School of Medicine, Aomori, Japan
b First Department of Internal Medicine, Hirosaki University School of Medicine, Aomori, Japan

Accepted for publication February 3, 2005.

* Address reprint requests to Dr Kondo, First Department of Surgery, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan (Email: h02gm501{at}cc.hirosaki-u.ac.jp).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: We tested whether administration of FK633, a short-acting glycoprotein IIb/IIIa inhibitor, before median sternotomy and cardiopulmonary bypass was able to interrupt the platelet activation loop and thereby preserve platelet number and function.

METHODS: This study investigated 16 pigs that underwent median sternotomy and 120 minutes of normothermic cardiopulmonary bypass (100 mL/kg) adding pericardial blood to the perfusate. FK633 was administered with heparin to one group (group F, n = 8), whereas only heparin was administered to the control group (group C, n = 8). Blood samples were obtained at several times, and complete blood count, platelet aggregation to adenosine diphosphate, thrombin-antithrombin complex, and bradykinin were evaluated. P-selectin expression and fibrinogen binding on platelet surfaces were measured by flow cytometry. Template bleeding times were measured before and after cardiopulmonary bypass. Chest tube drainage and hematocrit were determined at 2 and 6 hours after cardiopulmonary bypass.

RESULTS: In group F, platelet counts were preserved from 90 minutes of cardiopulmonary bypass. Platelet aggregation was inhibited at the beginning of cardiopulmonary bypass and showed no change at wound closure, and bleeding times were shortened at 2 hours after cardiopulmonary bypass. There were significant reductions in hematocrit of drainage. Flow cytometry showed no changes in P-selectin expression and fibrinogen binding in group F, whereas P-selectin expression and fibrinogen binding were elevated in group C.

CONCLUSIONS: Platelet inhibition with FK633 before invasive surgical procedure preserved platelet counts during and after cardiopulmonary bypass, and produced normal or near-normal bleeding times in the immediate postoperative period.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
In cardiac surgery, bleeding complications and excessive postoperative blood loss continue to contribute to the morbidity associated with cardiopulmonary bypass (CPB) [1, 2]. During CPB, blood components are exposed to artificial surfaces, and platelets are activated. This is followed by the release of various inflammatory cytokines and activation of the complement and coagulofibrinolytic systems [3], resulting in a decline in platelet number and function.

"Platelet anesthesia" is an attractive strategy for preserving platelet number and function that involves temporary pharmacologic inhibition of platelets during extracorporeal circulation [4–6]. Platelet aggregation is caused by activation of the glycoprotein (GP) IIb/IIIa complex and crosslinking with fibrinogen [7]. Studies with GP IIb/IIIa inhibitors such as tirofiban [4] and eptifibatide [5, 6] in a baboon CPB model revealed that these drugs preserved platelet number and shortened bleeding times after CPB. However, these studies were performed with peripheral cannulas, and therefore the animals did not undergo median sternotomy. Platelets are activated not only by contact with artificial surfaces and heparin administration [8] but also by the wound itself [9] and aspirated pericardial blood [10], and thus further confirmation is needed for the development of platelet anesthesia.

We used a porcine CPB model to test whether administration of FK633, a short-acting GP IIb/IIIa inhibitor [11, 12], can preserve platelets during and after CPB. FK633 was designed from the putative active structure of the Arg-Gly-Asp (RGD)-containing peptide and was synthesized at Fujisawa Pharmaceutical Co, Ltd. The drug has a half-life in plasma of 0.52 hours [11]. The drug is excreted mainly in the urine, mostly as unchanged compound [12]. Although FK633 is structurally an RGD peptide mimetic, its action is highly specific for GP IIb/IIIa [11]. Thus, FK633 is thought to be a specific antiplatelet agent without undesirable side effects [11], and in healthy human volunteers at therapeutic doses, FK633 produced no serious toxicologic or other adverse effects [12].

The key requirements for a platelet anesthetic are complete inhibition of all platelet function just before heparin is given and quick and complete reversal of this inhibition after CPB ends and protamine is given [4]. This reversal depends on elapsed time from discontinuation of GP IIb/IIIa inhibitors because these inhibitors have no neutralizing agent. Tirofiban and eptifibatide have half-lives in plasma of 1.6 hours [4] and 1 to 1.5 hours [5], respectively. Because FK633 has a shorter half-life than tirofiban and eptifibatide, FK633 is thought to be a promising GP IIb/IIIa inhibitor that can be used during CPB and reversed immediately after CPB. In a previous study, we reported platelet preservation in a simulated extracorporeal circulation circuit using FK633 while monitoring levels of ß-thromboglobulin [13]. FK633 inhibited platelet activation during simulated extracorporeal circulation and did not inhibit activated platelet consumption [13]. These results suggested that FK633 inhibits the amplification loop [14] by inhibiting the binding of fibrinogen to GP IIb/IIIa, thereby decreasing molecules that are released from activated platelets and generating new platelet activation. We hypothesized that FK633 administration before median sternotomy and CPB initiation interrupts this loop and preserves platelet number and function in an effective manner. We tested this hypothesis by measuring platelet number and function, thrombin generation, P-selectin expression, and fibrinogen binding on platelet surfaces using whole-blood flow cytometry [15].


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
The experiments reported herein were conducted in accordance with the Guidelines for Animal Experimentation of Hirosaki University.

Reagents
FK633 (N-{4-(4-amidinophenoxy)butyryl}-alpha-L-aspartyl-L-valine) was obtained from Fujisawa Pharmaceutical Co (Osaka, Japan). Fluorescein isothiocyanate-conjugated affinity-purified chicken anti-whole platelet antibody (09-128), anti-P-selectin antibody (09-143), and anti-fibrinogen antibody (09-038) were obtained from Immunsystem AB (Uppsala, Sweden). Adenosine diphosphate was supplied by MC Medical Co (Tokyo, Japan). Heparin sodium and protamine sulfate were supplied by Aventis Pharma Japan (Tokyo, Japan).

In Vitro Platelet Aggregation Study at Various Concentrations of FK633
Blood was withdrawn from pigs (n = 7) into tubes containing 1/10 volume of 3.8% sodium citrate and was then centrifuged at 100 g for 10 minutes before collection of platelet-rich plasma. The remaining blood was centrifuged for a further 10 minutes at 1,500 g to prepare platelet-poor plasma. The aggregation assay was performed using a laser light-scattering aggregometer (PA-200, Kowa, Tsukuba, Japan), with transmittance of platelet-poor plasma being calibrated as 100%. Platelet-rich plasma (240 µL) was incubated in the aggregometer with FK633 (30 µL, at final concentrations of 1, 3, 10, 30, 100, and 300 µmol/L) for 2 minutes at 37°C. Adenosine diphosphate (30 µL) was then added to a final concentration of 20 µM. Light transmittance was recorded, and percent inhibition was calculated based on aggregation of the control. FK633 dosage was determined from its molecular weight, porcine circulating blood volume, and the concentration of submaximum inhibition obtained from the dose-response data.

Porcine Cardiopulmonary Bypass Model
Sixteen pigs were used in this study. Anesthesia was induced with ketamine (30 mg/kg, intramuscular, Sankyo Co, Ltd, Tokyo, Japan) and pentobarbital sodium (5 mg/kg, intravenous, Dainippon Pharmaceutical Co, Ltd, Osaka, Japan). After anesthesia induction, pigs were placed in the supine position, and a tracheostomy was immediately performed. Pigs were then connected to the ventilator. Anesthesia was maintained by continuous intravenous infusion of midazolam (0.4 mg·kg–1·h–1, intravenous, Yamanouchi Pharmaceutical Co, Ltd, Tokyo, Japan), vecuronium bromide (0.06 mg·kg–1·h–1, Sankyo Co, Ltd), and ketamine (2 mg·kg–1·h–1). Hemodynamic monitoring was accomplished with a 16-gauge catheter placed in the femoral artery. The extracorporeal circuit consisted of polyvinyl chloride tubing, a venous reservoir and a membrane oxygenator (ExeLungPrime, HPO-20RHF, 2.4 m2 surface area, MERA, Inc, Tokyo, Japan), two polyurethane, wire-wrapped cannulas (MERA, Inc, Tokyo), and a roller head pump (Stockert Shiley, MERA, Inc). The circuit was primed with 500 mL of lactated Ringer’s solution, 300 mL of mannitol, and 200 mL of 6% hydroxyethylated starch. After median sternotomy, bilateral pleural opening and administration of 300 U/kg heparin, an arterial cannula (12F) was introduced into the right common carotid artery, and a two-staged venous cannula (28F) was introduced into the inferior vena cava by means of the right atrial appendage. These cannulas were connected with the circuit components. Normothermic CPB was initiated at 100 mL·kg–1·min–1 and was maintained for 120 minutes. Aspirated blood collected from mediastinal cavities and pleural cavities was added to the perfusate and recirculated. Pigs were divided into two groups: group C (n = 8), a control group; and group F (n = 8), which received bolus injection of FK633 (3 mg/kg before median sternotomy and 1.5 mg/kg immediately before heparin administration). Systemic heparinization was monitored by determination of activated coagulation time every 30 minutes, and activated coagulation time was maintained at a minimum of 400 seconds (normal value, 100 to 120 seconds) with a Hemocron 400 (Technidyne, Edison, NJ). Protamine (3 mg/kg) was given after removal of the cannulas. Protamine was added so as to maintain activated coagulation time at 100 to 120 seconds. A thoracic drainage tube was placed in the left pleural cavity, and was connected to the chest drainage bag (MD-8452S, Sumitomo Bakelite Co, Ltd, Tokyo, Japan) for continuous suction drainage at 10 cm H2O. Pigs were repositioned in the left lateral position after closure of median sternotomy and were observed until 6 hours after CPB termination.

Seven blood samples were obtained at anesthesia induction (baseline), at 2 minutes after heparinization, at 15 and 120 minutes after the start of CPB, immediately after wound closure, and at 2 and 6 hours after CPB termination. In addition to these points, complete blood counts were measured at 60 and 90 minutes after the start of CPB and at 4 hours after CPB termination. Blood samples were assayed for complete blood counts, platelet aggregation to adenosine diphosphate, bradykinin, thrombin-antithrombin complex (TAT), and flow cytometry. Bleeding times were measured following Duke’s method by making reproducible skin incisions on the upper lip with a lancet (blood lancet, Fether Safety Razor Co, Ltd, Osaka, Japan) at baseline, immediately after wound closure, and at 2 and 6 hours after CPB termination. Bleeding time measurements were duplicated and averaged. Chest tube drainage and its hematocrit were determined at 2 and 6 hours after CPB termination.

Measurements
Blood samples were obtained with ethylenediaminetetraacetic acid disodium (1.5 mg/mL blood) for complete blood counts, and with 3.8% sodium citrate for platelet aggregation and flow cytometry. Blood counts were measured using a fully automated blood cell analyzer (SF-3000 Sysmex, Kobe, Japan), and platelet aggregation was measured as described above. At each sampling point, blood was divided into collection tubes containing 3.8% sodium citrate for TAT or Trasylol, trypsin inhibitor, and protamine sulfate for bradykinin assay. Plasma samples were separated by centrifugation at 2,000 g for 10 minutes at 4°C after 15 minutes of cooling on ice. Plasma levels of TAT complex were measured using an enzyme immunoassay kit (SRL Inc, Tokyo, Japan) and levels of bradykinin were measured using a radioimmunoassay kit (SRL Inc). These assay procedures were performed according to the manufacturer’s protocols.

For flow cytometry, 5 µL of whole blood was added to polystyrene tubes containing 100 µL of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer (NaCl, 137 mmol/L; KCl, 2.7 mmol/L; MgCl2, 1 mmol/L; glucose, 5.6 mmol/L; bovine serum albumin, 1 g/L; HEPES, 20 mmol/L, pH 7.4) and 10 µL of fluorescein isothiocyanate-conjugated antibody. Samples were incubated for 10 minutes at room temperature and were then diluted and fixed with 1,000 µL of ice-cold phosphate-buffered saline (Na2HPO4, 0.02 mol/L; NaCl, 0.15 mol/L; NaN3, 0.02%, pH 7.2), containing 1% p-formaldehyde. No steps that induce platelet activation, such as washing, centrifugation, vortexing, or stirring, were used. Flow cytometry was performed using FACScan (Becton Dickinson, Tokyo, Japan). Data processing from 10,000 platelets was carried out with the Cell Quest (Becton Dickinson), an instrument that characterizes single platelets on the basis of cell size. The instrument measures the percentage of platelets demonstrating surface expression of P-selectin and fibrinogen binding. P-selectin-positive or fibrinogen-positive platelets were defined as having a fluorescence intensity exceeding that of 98% to 99% of negative control platelets.

All data are reported as mean ± standard deviation. Hemodilution was corrected by hematocrit in measurements of platelet counts, levels of TAT, and levels of bradykinin. The results of platelet counts and platelet aggregation are presented as a percentage of the baseline values for each point. Platelet counts are listed as percentages with or without hemodilution correction.

Statistical Analysis
We performed analysis using StatView for Windows J5.0 (SAS institute Inc, Cary, NC). Two-way analysis of variance for repeated measures was used for statistical analysis of group and time effects. The unpaired Student’s t statistic was used for specific comparisons at specific times between groups. The paired Student’s t test with Bonferroni correction was used for analysis of differences within groups. Differences were considered to be statistically significant when the probability value was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Effects of FK633 on Platelet Aggregation
The antiplatelet effects of FK633 were dose dependent (Fig 1). Inhibitory effects on platelet aggregation were 86.9% ± 2.7% and 87.9% ± 2.2% at final concentrations of 100 and 300 µmol/L, respectively. The amount of porcine circulating blood was estimated to be 65 mL/kg [16]. The molecular weight of FK633 was 454.48 [11]. We therefore decided to administer 3 mg/kg FK633 to the pig to give a final concentration of 100 µmol/L.



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  		Fig 1. Percent inhibition of platelet aggregation to adenosine diphosphate (ADP) by FK633. Error bars represent standard deviation of the mean. The FK633 dosage of 3 mg/kg was decided on the basis of molecular weight (454.48), porcine circulating blood volume (65 mL/kg), and concentration of submaximum inhibition (100 µmol/L).

 
Complete Blood Counts
At baseline, no significant differences in differential blood count were observed between the two groups: white blood cells (group F: 14.9 ± 3.7 versus group C: 16.6 ± 3.4; p = 0.18), red blood cells (F: 6.20 ± 0.88 versus C: 6.65 ± 0.58; p = 0.27), hemoglobin (F: 11.2 ± 1.5 versus C: 11.7 ± 0.8; p = 0.33), hematocrit (F: 37.3% ± 2.7% versus C: 35.9% ± 4.6%; p = 0.49), and platelets (F: 409 ± 91 versus C: 396 ± 82; p = 0.74). Throughout the course, these values did not differ significantly between the two groups, except for platelet count (hematocrit and platelet counts are listed Table 1, but other values are not shown). Platelet counts changed as a function of time, and the difference reached significance (Table 1). In group F, platelet counts increased above hematocrit recovery from dilution with the circuit contents, whereas platelet counts remained unchanged, despite hematocrit recovery in group C. In group F, platelet counts after 90 minutes of CPB were significantly higher than those in group C at the same sample time.


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  		Table 1. Efficacy Data at Each Timea
 
Platelet Aggregation to Adenosine Diphosphate Under In Vivo Conditions
Platelet aggregation was significantly lower in group F at heparin administration and at 15 minutes of CPB (Table 1). There was no significant difference at the end of CPB.

Bleeding Times
Bleeding times were significantly longer immediately after wound closure in both groups when compared with baseline values (Table 1, Fig 2). In group F, bleeding times returned to baseline value within 2 hours of CPB, but bleeding times in group C returned incrementally.



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  		Fig 2. Bleeding times. Diamonds and the continuous line represent the control group (group C); triangles and the dotted line represent the FK633-treated group (group F). Error bars represent standard deviation of the mean. *p < 0.05 between baseline and subsequent time points (paired Student’s t test with Bonferroni correction). {dagger}p < 0.05 between group F and group C at specific time points (unpaired Student’s t test with Bonferroni correction). (Base = baseline; Hep = after heparin and FK633 administration before cardiopulmonary bypass [CPB]; Closure = immediately after wound closure; 2 and 6 = hours after CPB termination.)

 
Flow Cytometry
In group F, the percentage of platelets exhibiting P-selectin expression remained unchanged throughout extracorporeal circulation, whereas that in group C increased significantly after 15 minutes of CPB (Table 1). P-selectin-positive platelets increased at 2 and 6 hours after CPB in both groups. Fibrinogen-positive platelets changed in a similar manner as those expressing P-selectin.

Soluble Markers
The levels of bradykinin in group F were lower after CPB, but the difference did not reach significance (Table 1). In both groups, TAT levels increased during CPB and decreased after CPB in a similar manner.

Volume and Hematocrit of Chest Tube Drainage
There were significant differences in the hematocrit of chest tube drainage at 2 hours after CPB (F versus C, p = 0.022) and at 6 hours after CPB (F versus C, p = 0.022). Chest tube drainage was less in group F at 2 hours after CPB (F versus C, p = 0.082) and at 6 hours after CPB (F versus C, p = 0.150), but this difference was not significant.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Glycoprotein IIb/IIIa inhibitors given to patients with acute coronary syndrome modified the clinical outcome of these patients [17]. After treatment with abciximab, the median number of transfusions in patients undergoing urgent coronary artery bypass grafting surgery was significantly higher [17]. This was attributed to a prolonged and irreversible antiplatelet effect [18]. Bizzarri and colleagues [1] reported that infusion of tirofiban immediately before surgical intervention did not adversely affect clinical outcome and maintained hemoglobin and platelet counts at stable levels. Although this study is considered to be collateral evidence for previous animal data [4], tirofiban is not yet used during CPB. FK633 has a reversible antiplatelet effect that is shorter than that of tirofiban, and thus FK633 can be actively used during surgery. We developed a porcine CPB model to test whether infusion of FK633 during CPB was able to preserve platelet counts and decrease blood loss under invasive conditions with median sternotomy and addition of blood aspirated from the pericardial cavity to the perfusate. Pig organs and platelets share anatomic and physiologic similarities with those of humans [19, 20]. For this reason, pigs are often used in experimental research on platelets [21, 22] or in extracorporeal circulation models [9, 23]. The first dose is intended to inhibit platelet activation induced by median sternotomy [9]. Additional doses are intended to inhibit platelet activation resulting from heparin administration [8], blood exposure to artificial surfaces [3], and aspirated blood collected from mediastinal cavities and pleural cavities [10]. This protocol preserved platelet counts and accelerated recovery of bleeding times in group F. Platelet counts in group F increased during CPB and exceeded baseline values, after correction for hemodilution. These increases in platelet count appeared to be the result of new platelet entrance into systemic circulation from the bone marrow and spleen [24, 25]. Platelet counts did not increase either during or after CPB in group C. Platelet consumption might have exceeded new platelet arrival.

Platelet aggregation was inhibited by FK633 at heparin administration, which was an anticipated result from preliminary data, and recovered to levels comparable with group C at the end of CPB. The platelet population was heterogeneous at the end of CPB [24]. Some platelets were newly arrived from the bone marrow, but most were partially degranulated and had damaged membranes [24] or decreased numbers of fibrinogen receptors [25]. Platelet function is also problematic after CPB, but temporary inhibition of platelets during CPB can reduce platelet damage [24]. The results of flow cytometry confirmed the inhibition of platelet activation during CPB and the reversal of this inhibition. Platelet preservation with FK633 thus resulted in shortened template bleeding times and decreased blood loss.

The term platelet anesthesia should be used when an inhibitor or protocol achieves a return of overall platelet function to control values at the time protamine is given. The present study was not able to demonstrate conclusive results regarding this point. In group F, bleeding time did not return to control values at wound closure. In a previous study with tirofiban, bleeding times remained prolonged during the first few hours after surgery [4]. Although eptifibatide alone significantly shortened postoperative bleeding times at the time of protamine administration, bleeding times were not restored to preoperative values until 60 minutes after protamine administration [5]. However, the combination of eptifibatide and nitric oxide restored preoperative bleeding times at the time of protamine administration [6]. Nitric oxide has a half-life of less than 1 second in blood, and its effects may be limited to the oxygenator and arterial line of the CPB circuit [6]. The combination of FK633 with other inhibitors, such as nitric oxide, might yield a more rapid restoration of platelet function. Further studies are necessary to define a therapeutic protocol for using FK633.

We also measured plasma levels of soluble markers reflecting activation of the coagulation system. Anticoagulation with heparin during CPB does not entirely prevent surface-induced coagulation and adherence of blood cells to membranes [26]. Contact with the artificial surfaces of the extracorporeal circuit activates the intrinsic coagulation pathway in which bradykinin is produced by the kallikrein-kinin system [23]. The activated coagulation pathway generates thrombin, and thrombin causes extrinsic coagulation activation through tissue factor and factor VIIa [27]. Platelets play a major role in blood coagulation, and several of the key enzymatic reactions of the coagulation cascade occur on the platelet membrane [28]. It is therefore reasonable to postulate that pharmacologic inhibition of platelets may affect coagulation mechanisms and thrombin generation. Byzova and Plow [29] reported that GP IIb/IIIa binds to pr othrombin and accelerates prothrombin activation, and that GP IIb/IIIa antagonists inhibit thrombin generation by influencing platelet-coagulant protein interactions. Levels of TAT complex are indicative of plasma thrombin levels induced by the activated coagulation system. Rao and associates [28] reported that tirofiban inhibited not only platelet aggregation but also thrombin generation using a baboon CPB model [4]. Although low-dose and high-dose infusion decreased TAT levels, those in the high-dose group were lower than in the low-dose group and were significantly different from those in the control group [28]. The present study did not show significant differences in intrinsic coagulation pathway activation or thrombin formation. Higher dose administration of GP IIb/IIIa inhibitor might be needed to have an effect on thrombin generation in addition to the platelet preservation observed during CPB. This model is limited in its ability to evaluate tissue factor and extrinsic pathways that potentially interact with GP IIb/IIIa inhibitors because of the lack of an ischemia-reperfusion process, which triggers tissue factor and thrombin generation [27]. This model should also be developed to investigate the interaction between platelets and the coagulation pathway.

In conclusion, FK633, a short-acting GP IIb/IIIa inhibitor, preserved platelet counts by inhibiting platelet activation during CPB and produced normal or near-normal bleeding times during the immediate postoperative period under invasive in vivo conditions.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
The authors thank Yuko Tsushima for performing flow cytometry, Masakatsu Nakagawara (MERA, Inc, Tokyo, Japan) for creating the perfusion circuits and supervising CPB, and Shigeyuki Nakaji, MD, PhD, for his assistance with statistical analysis.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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Invited commentary

Ann. Thorac. Surg. 80: 257-258. [Full Text]




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