Eur J Cardiothorac Surg 2004;26:289-293
© 2004 Elsevier Science NL
Efficacy of FK633, an ultra-short acting glycoprotein IIb/IIIa antagonist on platelet preservation during and after cardiopulmonary bypass
Shigeki Tabata*,
Syohjiro Yamaguchi,
Hiroshi Nagamine,
Shigeyuki Tomita,
Sadahiko Arai,
Hirofumi Takemura,
Go Watanabe
Department of General and Cardiothoracic Surgery, Kanazawa University School of Medicine, Takaramachi 13-1, Kanazawa 920-8641, Japan
Received 18 September 2003;
received in revised form 11 February 2004;
accepted 8 March 2004.
* Corresponding author. Tel.: +81-76-265-2354; fax: +81-76-222-6833
e-mail: 02117tabata{at}kouseiren-ta.or.jp
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Abstract
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Objective: Temporary pharmacologic inhibition of platelet function during and after cardiopulmonary bypass (CPB) (platelet anesthesia) is an attractive strategy for preserving platelets during CPB. We examined the efficacy of FK633, an ultra-short acting glycoprotein IIb/IIIa antagonist. Methods: The study was carried out in six mongrel dogs that received an intravenous bolus of 0.1 mg/kg of FK633 at the time of administration of heparin (group F), and six control dogs (group C). All animals underwent 60 min of normothermic CPB followed by a 2-h observation period. Blood samples for platelet count, platelet aggregation to adenosine diphosphate and parameters concerning the coagulation system were obtained at eight time points. Hemodynamics, bleeding time, and postoperative blood loss were assessed serially. Scanning electron micrograph of the oxygenator's membrane was investigated. Results: FK633 significantly protected platelet number (group F, 59±10% versus group C, 38±15% of the pre-CPB value; P<0.01), and inhibited platelet aggregation to adenosine diphosphate (group F, 13±12% versus group C, 35±9% of the pre-CPB value; P<0.01) during CPB. Postoperative blood loss did not significantly differ between the two groups, but there was a tendency of less bleeding in group F (group F, 73±23 ml versus group C, 111±44 ml; P=0.09). In group F, scanning electron micrograph of the oxygenator's membrane showed that its surface was free from platelets. There were no significant differences between the groups in hemodynamics. Conclusions: An ultra-short acting glycoprotein IIb/IIIa antagonist, FK633, is effective in preventing both platelet aggregation and thrombocytopenia during CPB, and may be effective for minimizing postoperative bleeding.
Key Words: Cardiopulmonary bypass • Platelet • Glycoprotein IIb/IIIa antagonist • Platelet anesthesia
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1. Introduction
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The use of cardiopulmonary bypass (CPB) for open heart surgery is associated with a generalized coagulopathy [1]. Bleeding abnormalities after CPB are related to heparin usage, fibrinolysis, platelet loss, and a combination of these factors, and the decrease in platelet numbers and function is believed to be the most significant cause [2]. Platelets are decreased, destroyed, and altered during CPB because of adhesion to the surfaces of the CPB circuitry, activation and aggregation.
Temporary pharmacologic inhibition of platelet adhesion and activation during CPB is an attractive strategy to preserve platelet number and function. This concept of ‘platelet anesthesia’ [3] has been tried with a variety of reversible platelet inhibitors, such as prostaglangin E1, dipyridamole, and desmopressin [4–7], but none of them were effective enough to be satisfactory inhibitors. Ideally, the inhibitor must disappear or be completely reversed at the time protamine is given.
Interaction between platelet glycoprotein (GP) IIb/IIIa and fibrinogen is involved in platelet adhesion and aggregation during CPB [8]. However, none of them could be reversed quickly enough to achieve normal bleeding time when heparin was reversed by the administration of protamine after CPB. Residual inhibition of platelets by the GP IIb/IIIa antagonists were probably responsible for the prolonged bleeding time.
It has been tested that several GP IIb/IIIa antagonists can provide ‘platelet anesthesia’ [3,9–11]. This study examined the efficacy of an ultra-short acting GP IIb/IIIa antagonist, FK633 (N-{4-(4-amidinophenoxy)butyryl}-
-L-aspartyl-L-valine; Fujisawa Pharmaceutical Company, Tokyo, Japan), in preserving both platelet number and function during CPB in a canine model. The drug is a new selective nonpeptide GP IIb/IIIa antagonist, and has a half-life in plasma of 31.2 min [12]. We conducted this study under the assumption that, with such a short half-life, FK633 would be able to sufficiently provide ‘platelet anesthesia’ during CPB and then be adequately reversed.
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2. Material and methods
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2.1. Experimental study
Twelve adult mongrel dogs weighing 14.5±4.5 kg (range 9.7–21.0 kg) were studied. The dogs received humane care in compliance with the ‘Principles of Laboratory Animal Care’ (National Society for Medical Research) and the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (NIH published 85-23, revised 1985). The animals were anesthetized with an intramuscular administration of ketamine hydrochloride (20 mg/kg), and intravenous sodium pentobarbiturate (30 mg/kg), and were mechanically ventilated with a volume respirator. A catheter inserted through the right femoral artery was used to measure systemic blood pressure and obtain blood samples. A Swan-Ganz catheter (American Edwards Laboratories, Santa Ana, CA, USA) introduced through the right femoral vein was used to measure pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and cardiac output.
A right parasternal incision was made, extending from the lower edge of the second rib to the superior edge of the fifth rib. Six dogs were given FK633 (group F) as an intravenous bolus of 0.1 mg/kg, and six served as controls (group C). After systemic heparinization (200 U/kg intravenously), CPB was initiated. CPB was achieved with a centrifugal pump (Capiox, Terumo Corporation, Tokyo, Japan) and a membrane oxygenator (Capiox Hollow Fiber Oxygenator, Terumo Corporation, Tokyo, Japan). The priming volume of the circuit was 300 ml of lactated Ringer's solution, hydroxyethylated starch, D-mannitol, sodium bicarbonate, and heparin sodium (100 U/kg). During CPB, the flow rate was kept at 90 ml/kg per min and the heart continued beating. The mean arterial pressure was kept at 60–80 mmHg and the systemic temperature was maintained at 37 °C. After 60 min of CPB, all animals were weaned from CPB. Protamine sulfate (1.5 mg/1000 U heparin) was given after removing the cannulae. The animals were observed under general anesthesia for 2 h after CPB.
Blood samples were obtained at the following time points: baseline before the administration of heparin and platelet inhibitors (time point expressed as Base); 5 min after the administration of heparin and platelet inhibitors (Heparin); 5, 30, and 60 min after initiation of CPB (On, 30 min, Off, respectively); and 10, 60 and 120 min after the administration of protamine (Protamine, P-60, P-120, respectively). Bleeding times were obtained at baseline before the administration of heparin and platelet inhibitors, and 10, 60 and 120 min after the administration of protamine.
Blood samples were assayed for hematocrit, platelet count, and adenosine diphosphate (ADP)-induced platelet aggregation.
Heart rate by electrocardiogram, systemic arterial pressures, central venous pressure, pulmonary arterial pressure, and pulmonary capillary wedge pressure were continuously monitored.
2.2. Measurements
Platelet counts were corrected with hematocrit and were expressed as a percentage of baseline values. Platelet aggregation studies were performed on platelet rich plasma in the presence of 100 µmol/l ADP. Inhibition and recovery of platelet aggregation was expressed as a percentage of baseline values.
Bleeding time was assessed by making a standard skin incision on the inside of the ear, absorbing blood onto filter paper, and noting the time until cessation of bleeding.
After protamine administration and during the observation period, thoracic blood loss was determined hourly by aspiration of the blood collected in the chest cavity.
At the end of the experiment, the oxygenator was separated from the circulation system and was rinsed with saline. Pieces measuring approximately 1 cm2 were cut from the membrane. These specimens were fixed for 60 min at 4 °C with 2.5% glutaraldehyde buffered with sodium cacodylate, 0.1 mol/l, at pH 7.4. After, they were rinsed with the same buffer, the specimens were dehydrated in acetone and dried at the critical point of carbon dioxide. Dried samples were mounted with a double-sided adhesion tape on scanning electron microscope specimen holders and were sputter-coated with gold (15 nmol). The specimens were then examined with a scanning electron microscope (JSM-5400, Jeol Ltd, Tokyo, Japan).
2.3. Statistical analysis
Results are expressed as mean±standard deviation. Two-way analysis of variance for repeated measures with Dunnett's test was used for statistical analysis of group and time effects. Blood loss was compared using Student's t test. Results were considered statistically significant at P<0.05.
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3. Results
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No significant differences between the two groups were observed in cardiac output, heart rate, arterial/central venous pressures, or pulmonary arterial/pulmonary capillary wedge pressures, and all values remained within normal ranges at all time points.
At the end of CPB, group F had significantly higher platelet counts, when compared with those in group C (59±10 versus 38±15%, respectively; P<0.01). Two hours after CPB, platelet counts were better preserved in group F (83±10 versus 58±22%, respectively; P<0.01) (Fig. 1)
. ADP-induced platelet aggregation expressed as a percent of the pre-CPB value was significantly inhibited in group F 30 min into CPB (13±12 versus 35±9%, respectively; P<0.01). Platelet aggregation in group F was recovered 2 h after CPB (65±17 versus 79±13%, respectively; NS) (Fig. 2)
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Fig. 1. Changes in hematocrit-corrected platelet counts. Platelet counts are normalized for the baseline value at each time point. *P<0.01 versus the control group, by repeated-measures analysis of variance. Base (before administration of heparin and GP IIb/IIIa antagonists); Heparin (5 min after administration of heparin and GP IIb/IIIa antagonists before cardiopulmonary bypass (CPB)); On (5 min after the start of CPB); 30 min (30 min after the start of CPB); Off (60 min after the start of CPB); Protamine (10 min after administration of protamine); P-60 and P-120 (60 and 120 min after administration of protamine).  , group C; •, group F. Each value represented as mean±standard deviation.
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Fig. 2. Changes in platelet aggregation induced by ADP. Data are normalized for the baseline value.
P<0.05 versus the control group, *P<0.01 versus the control group, by repeated-measures analysis of variance. Abbreviations and symbols are as in Fig. 1. Each value represented as mean±standard deviation.
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After CPB, bleeding times increased in both groups, but in group F bleeding time at 1 h after protamine administration did not significantly differ from that obtained at baseline (Fig. 3)
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Postoperative blood loss did not significantly differ between the two groups, but there was a tendency of less bleeding in group F (73±23 versus 111±44 ml, respectively; P=0.09) (Fig. 4)
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Scanning electron micrograph of the oxygenator membrane of a dog from group C showed that its surface was covered with adherent platelets (Fig. 5)
. In contrast, platelet aggregates on the oxygenator membrane were absent in group F (Fig. 6)
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Fig. 5. Scanning electron micrograph of the oxygenator's membrane. Photomicrograph of a control dog without FK633 treatment. The outer surface of the membrane is covered with adherent platelets.
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Fig. 6. Scanning electron micrograph of the oxygenator's membrane. In a dog treated with FK633, the surface of the oxygenator's membrane is free from platelets.
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4. Discussion
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CPB is often associated with severe complications such as hematologic disorders [1] and generation of a systemic inflammatory response [13]. Bleeding complications after CPB are related to heparin usage, fibrinolysis, platelet loss, and a combination of these factors, and it is believed that the most significant, yet unsolved cause is the decrease in platelet number and function [2].
During CPB, platelets are partially activated by heparin [14], and are diluted, destroyed [2], activated when adhered to foreign surfaces [15], and form aggregates [16]. By the end of CPB, platelet count and function is reduced, and bleeding time is prolonged. In addition, formation of microthrombi could be responsible for end organ damage [17].
‘Platelet anesthesia’ is a concept to prevent thrombocytopenia following CPB by temporary pharmacologic inhibition of platelet adhesion and activation during CPB [3]. After this concept was introduced with the use of prostaglandin E1 [4], a variety of platelet inhibitors have been tried. Reversibility must be achieved by instant clearance of the drug from the blood, because there are no known antidotes to platelet inhibitors. Dipyridamole, phosphodiesterase inhibitor, only partially inhibits platelets during CPB [5], and is not quickly reversible because of a long plasma half-life (100 min). Prostanoids, particularly the prostacyclin analog "iloprost", incompletely inhibit platelet membrane receptors and are reversible, but cause profound and unacceptable hypotension [6]. Desmopressin [7] prevents platelet consumption in vitro but fails to preserve platelets in vivo.
The GP IIb/IIIa complex is a major constituent of the platelet membrane and is known to play a key role in the interaction between platelets and fibrinogen adsorbed on the foreign surfaces of the CPB tubing and oxygenator [11]. It has been tested that several GP IIb/IIIa antagonists can provide ‘platelet anesthesia’ [3,9–11], but platelet restoration could not be achieved rapidly enough after CPB. In this study, we examined the efficacy of an ultra-short acting antagonist, FK633 in a canine model. The drug is a new selective nonpeptide GP IIb/IIIa antagonist, and has a half-life in plasma of 31.2 min [12]. In dogs, bolus injection of FK633 at 0.1 mg/kg significantly suppressed ex vivo ADP-induced platelet aggregation (>40% inhibition) and thrombus formation at stenosed and injured coronary arteries, but did not prolong bleeding time [12].
In our study, an ultra-short acting GP IIb/IIIa antagonist prevented not only platelet aggregation but also the loss of platelet number during CPB, which resulted in less postoperative bleeding at an experimental level. Moreover, in group F, platelet aggregates were absent on the scanning electron micrograph of the oxygenator's membrane (Fig. 6). This finding suggests that FK633 can prevent destruction of platelets on the oxygenator's membrane, which results in the preservation of platelet number and function. On the other hand, FK633 was not able to prevent the initial drop in platelet count. Furthermore, in animals that received FK633, platelet aggregation did not return to control values until 2 h after CPB (Fig. 2). Further studies are necessary to define the therapeutic dose of FK633. In addition, a combination with other inhibitors such as Iloprost, in low doses insufficient to cause vasodilatory side effects [18], may be effective in allowing a more rapid restoration of platelet function.
The key requirements for a platelet anesthetic are complete inhibition of all platelet functions just before heparin is given and quick and complete reversal of this inhibition when protamine is given after CPB [3]. Further studies are required to show a safe therapeutic index for FK633 and to confirm the absence of side effects in the clinical situation.
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Y. Hiramatsu
Invited commentary
Ann. Thorac. Surg.,
July 1, 2005;
80(1):
257 - 258.
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Abstract
1. Introduction
, group F; 
, group C; bar, standard deviation.