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ORIGINAL ARTICLE |
Department of Cardiovascular Surgery, Tokyo Women’s Medical University, Tokyo, Japan
Toru Okamura, MD, Tel: +81 3 3353 8111, Fax: +81 3 3353 4472, Email: tokamura{at}hotmail.co.jp, Department of Cardiovascular Surgery, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjyuku, Tokyo 162-8666, Japan.
ABSTRACT
Heparin is the routine anticoagulant for cardiopulmonary bypass, but complications due to heparin are often reported. This study assessed argatroban as an alternative to heparin. Normothermic cardiopulmonary bypass with hemodilution was performed for 2 h in 15 dogs (mean weight, 9.8 kg) randomly assigned to 3 groups of 5 each. The controls were given heparin 200 IU·kg–1 before cardiopulmonary bypass; group A had argatroban infused continuously at a rate of 20 µg·kg–1·min–1; group H/A had half doses of both heparin (100 IU·kg–1) and argatroban (10 µg·kg–1·min–1). Blood samples were collected at 5 time points during the experiment. Activated clotting time, hemoglobin level, platelet counts, and serum concentrations of fibrinogen, antithrombin III, and thrombin-antithrombin III complex were measured. The platelet count was reduced significantly, and the production of thrombin-antithrombin III complex was inhibited in group H/A. Activated clotting time remained <300 sec at all time points in group A, but it was maintained at approximately 400 sec in group H/A. Fibrinogen and antithrombin III levels were reduced to half in all groups after initiation of cardiopulmonary bypass. The simultaneous use of heparin and argatroban infusion might be useful for cardiopulmonary bypass with hemodilution.
Key Words: Anticoagulants • Argatroban [Substance Name] • Cardiopulmonary Bypass • Heparin
INTRODUCTION
The technique, apparatus, and understanding of extracorporeal circulation have advanced remarkably over the past decades. Nevertheless, despite efforts to improve the biocompatibility of extracorporeal circuits, activation of inflammatory pathways, including coagulation and fibrinolysis, remains a major problem. Activation of inflammatory pathways by cardiopulmonary bypass (CPB) can result in perioperative multiple organ dysfunction and bleeding syndromes, which are not uncommon complications of CPB. Heparin is routinely used as an anticoagulant because of its reversibility, cost, and relative safety for heart surgery under CPB. However, heparin anticoagulation is catalyzed by antithrombin III (AT-III), and several consequences have emerged, including heparin-induced thrombocytopenia, AT-III deficiency, and aggregation of platelets. In addition, clot-bound thrombin is not inhibited effectively by the heparin-AT complex, and platelet activation and fibrin formation may still occur.1–4 Inadequate thrombin suppression results in excessive consumption of plasma coagulation factors and platelets, and microvascular bleeding may follow after cardiac surgery. These reactions occur more strongly in neonates and infants because of their immature organs and low body weight. Recently, avoidance of blood infusion during cardiac surgery was proposed to reduce infection risks, but autologous blood alone during CPB might severely decrease the hematocrit, especially in children with low body weights. At a result, oxygen delivery and choroid pressure would decrease. Moreover, numerous proteins in blood, including antithrombin III, would be reduced. As heparin requires antithrombin III, we attempted to find another anticoagulant protocol for CPB with hemodilution. Argatroban, a derivative of arginine, is a direct selective thrombin inhibitor with a half-life of 15–30 min. Argatroban binds reversibly to thrombin without AT-III, and develops effective anticoagulant activity. However, the efficacy of argatroban for anticoagulation during CPB is controversial. We hypothesized that argatroban might be effective as a substitute for heparin and as an adjunct to heparin anticoagulation. This was tested in an animal model of CPB.
MATERIALS AND METHODS
All animals involved in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised in 1996. Fifteen beagle dogs, with a mean weight of 9.8 kg, were anesthetized by intramuscular injection of 10 mg·kg–1 sodium pentobarbital. An intravenous line was inserted, and sodium pentobarbital 25 mg·kg–1 and pancuronium bromide 0.08 mg·kg–1 were infused after endotracheal intubation and establishing artificial ventilation with a pressure-controlled respirator. A median sternotomy was performed, and CPB was established with cannulation of the ascending aorta and right atrial appendage. The CPB circuit was made of standard uncoated vinyl chloride tubing (without a suction circuit), an open hard shell reservoir, and an artificial membrane oxygenator with a heat exchanger (Lilliput 1; Dideco-Sorin, Puchheim, Germany). The priming solution for the circuit consisted of lactated Ringer’s solution (400 mL), sodium bicarbonate 7.4% (50 mL), and mannitol (50 mL). CPB was performed with non-pulsatile flow (Sarns pump; Terumo, Tokyo, Japan) at a rate of 80–100 mL·kg–1·min–1 at 37°C for 2 h.
The 15 dogs were randomly divided into 3 groups of 5 each: group H (control) had heparin 200 IU·kg–1 infused 5 min before start of CPB; group A had argatroban (Mitubishi Tanabe Pharma, Osaka, Japan) 20 µg·kg–1·min–1 infused 10 min before start of CPB and maintained at this rate during CPB; and group H/A had a half dose of argatroban (10 µg·kg–1·min–1) infused from 10 min before CPB and a half dose of heparin (100 IU·kg–1) injected 5 min before the start of CPB.
Blood samples were obtained at baseline (before infusion of drug) and at 10, 60, 90, and 120 min after initiation of CPB. Activated clotting time (ACT), hemoglobin, platelet count, fibrinogen, AT-III, and thrombin-AT complex (TAT) were measured. The ACT was measured using a Hemochron 401 whole blood detection system (International Technidyne Co., Edison, NJ, USA) with Celite ACT tubes (International Technidyne). Hemoglobin and platelet counts were determined using an automated analyzer (Coulter Electronics, Harpenden, UK). AT-III was measured by chromogenic assay. Fibrinogen was determined by the Clauss thrombin time method. TAT was measured by time-resolved fluorescence immunoassay. Blood samples for TAT and fibrinogen were immediately cooled on ice and centrifuged, and the plasma was stored at –70°C before being measured with an assay kit (SRL; Tokyo Medical, Tokyo, Japan).
All quantitative data are expressed as the mean ± standard error. Repeated measures analysis of variance (ANOVA) was used to evaluate changes over time and to compare rates of change among the groups. One-way ANOVA with Bonferroni t tests were used to detect differences in platelet counts, TAT, AT-III, and fibrinogen among the groups. A p value less than 0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 12.0 (SPSS, Inc., Chicago, IL, USA).
RESULTS
The ACT was maintained at approximately 400 sec in group H/A at all time points. The ACT during CPB was significantly lower in group A than in the other groups; ACT in group A was <300 sec at all time points (Figure 1
). Hemoglobin values were reduced in all groups just after the start of CPB, due to hemodilution by the priming solution. There was no significant difference in hemoglobin values among the 3 groups (Figure 2A). Platelet counts decreased after initiating CPB in all groups, and there were no significant differences at baseline. However, the platelet counts in group H/A were significantly higher than those in the other groups (p <0.05) after 120 min of CPB (Figure 2B). The plasma levels of fibrinogen and AT-III decreased after the initiation of CPB in all groups; there were no significant differences among groups (Figure 3). The plasma levels of TAT were significantly lower in group H/A than in the controls (p = 0.03) and group A (p = 0.001), as shown in Figure 4.
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Several investigators have suggested that retransfused suctioned blood from the pericardial cavity could be the major source of a thrombin-generating agent.5,6 More recently, Philippou and colleagues7 demonstrated that pericardium-induced activation of factor VII, due to ineffective local heparinization, results in increased thrombin generation. Therefore, regimens that preserve the coagulation and fibrinolysis systems during and after CPB are needed. Hirudin, bivalirudin, and argatroban are currently approved by the US Food and Drug Administration as direct thrombin inhibitors.8
Hirudin is a bivalent inhibitor of thrombin. The terminal half-life of hirudin is approximately 60 min in healthy volunteers. Hirudin is cleared via the kidneys, and accumulates in patients with renal insufficiency; no specific antidote is available to reverse its anticoagulant effect. Bivalirudin is synthetic version of hirudin, with a half-life of approximately 25 min. Renal excretion is not the major route of bivalirudin clearance: it is degraded by endogenous peptidases. Argatroban is a small molecule (molecular weight, 527) that acts as a competitive inhibitor of thrombin and is metabolized by the liver. Argatroban has an advantage over other drugs in this metabolic pathway for a patient with heart disease, because renal dysfunction does not affect its plasma half-life.
Active-site-directed inhibitors, such as argatroban, inhibit fibrin-bound thrombin without displacing the enzyme from fibrin, whereas bivalent inhibitors, such as hirudin or bivalirudin, displace thrombin from fibrin during the inactivation process.8,9 Unlike heparin, direct thrombin inhibitors bind reversibly to thrombin without the need for AT-III to induce anticoagulant activity, and they prevent decreased platelet counts by inhibiting thrombin-induced platelet aggregation.10,11 On the other hand, heparin binds to plasma proteins, some of which are acute phase reactants and others are released from platelets or endothelial cells activated by the products of clotting, and the anticoagulant response to heparin varies among patients.12,13 Argatroban has been used successfully in patients with renal failure and AT-III deficiency during hemodialysis.14 Samuels and colleagues15 concluded that argatroban alone or simultaneously with heparin serves as an adequate postoperative anticoagulant regimen in patients with ventricular assist devices. We considered it necessary to establish a regimen for argatroban use during CPB. In this animal model, it was obvious that argatroban alone was not effective during CPB, but simultaneous infusion of argatroban with heparin pretreatment preserved the platelet count and reduced the production of TAT. These results suggest that the combination of argatroban and heparin inhibits the activation of platelets and the coagulation cascade during CPB. Argatroban may also be effective in inhibiting clot-bound thrombin which is resistant to neutralization by the heparin-AT complex (mean molecular weight of heparin, 15,000). The combination of argatroban and heparin may have advantages in overcoming AT-III deficiency and inhibiting the catalytic activity of clot-bound thrombin.16
The ACT is employed to evaluate the anticoagulant effect of heparin, and it is maintained at 400 sec or more during CPB.17 Sakai and colleagues18 reported that the ACT was not controlled >400 sec using an argatroban dose of 2 mg before CPB and 10 µg·kg–1·min–1 during CPB. Therefore, to try to control the ACT at >400 sec, we selected 2 mg of argatroban to infuse before CPB and 20 µg·kg–1·min–1 during CPB. Rikitake and colleagues19 reported that the ACT could not be controlled >400 sec with an argatroban infusion of 30 µg·kg–1·min–1 in a dog model. They also observed no thrombus in the arterial filter of the CPB circuit or in the kidneys and spleen. However, using a similar protocol, we could not maintain the ACT >400 sec for the duration of CPB, but in group H/A, the ACT could be maintained at approximately 400 sec. This may indicate an additive effect of argatroban and heparin. It might be possible to decrease the amount of heparin during CPB using this regimen. Bleeding after CPB might be reduced by avoiding excessive heparin. Kawada and colleagues20 reported the first clinical use of argatroban as an anticoagulant during left heart bypass in surgery for aortic aneurysm. We suggest that the development of a clinically appropriate protocol for the administration of argatroban during CPB needs further investigation.
Some limitations should be considered in this study. We did not use suction to remove blood from the surgical field during CPB; therefore, thrombogenicity, including the activation of platelets and the coagulation cascade, might be reduced much less in our model than in actual cardiac surgery. In addition, differences between human and dog coagulation systems should be considered. However, the simultaneous infusion of both argatroban and heparin may be useful in cardiac surgery with CPB. An anticoagulant protocol with argatroban alone during CPB may not be an adequate substitute for heparin. Further investigations are needed to resolve the heparin and argatroban interactions.
REFERENCES
Asian Cardiovasc Thorac Ann 2010;
18:22-26
© 2010 by SAGE Publications
DOI: 10.1177/0218492309352034
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