Ann Thorac Surg 2000;70:186-190
© 2000 The Society of Thoracic Surgeons
Original articles: Cardiovascular
Reduction of heparin dose is not beneficial to platelet function
Takayuki Nakajima, MDa,
Kohei Kawazoe, MDa,
Kazuyuki Ishibashi, MDa,
Yoshimitsu Kubota, CCPa,
Tatsuya Sasaki, MDa,
Hiroshi Izumoto, MDa,
Tohru Nitatori, PhDb
a Third Department of Surgery, Iwate Medical University School of Medicine, Iwate, Japan
b Department of Cell Biology and Neuroanatomy, Iwate Medical University School of Medicine, Iwate, Japan
Address reprint requests to Dr Nakajima, Third Department of Surgery, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, Iwate, 020-8505, Japan
e-mail: t_nakajima{at}imu.ncvc.go.jp
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Abstract
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Background. To clarify the effects of the reduction of heparin dose on platelets, we conducted a prospective trial on patients undergoing cardiopulmonary bypass.
Methods. Twenty-three patients undergoing coronary artery bypass grafting were studied. The systemic heparin dose was 300 IU/kg in the control group ( 
) and 200 IU/kg in the low-dose group ( 
). Heparin-coated cardiopulmonary bypass equipment was used for both the groups. Platelet counts, ß-thromboglobulin (ß-TG) and platelet factor 4 (PF4) concentrations were measured and the arterial filters in the circuits were observed by electron microscopy.
Results. Platelet counts were higher in the low-dose group than in the control group (p < 0.01). No significant differences were found in the platelet release reaction (ß-TG and PF4). Electron microscopy demonstrated that cell adhesion on the arterial filters in the control group was significantly more marked than in the low-dose group (p < 0.01) and that most of the cells on the filters were neutrophils.
Conclusions. We conclude that the reduction of heparin dose with the use of heparin-coated equipment reduces platelet loss, but does not suppress the platelet release reaction. Furthermore, the reduction of heparin dose reduces adherence of leukocytes to the filter surface.
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Introduction
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Cardiopulmonary bypass (CPB) induces thrombocytopenia, formation of platelet aggregates, and release of platelet granule contents, resulting in excessive blood loss. Platelet dysfunction during CPB is known to be induced by consumption in the body, blood–gas interactions, hypothermia, systemic heparinization, and adhesion to the circuits. Therefore, a number of approaches to preserve platelet function have been attempted. One of these is a reduction of heparin dose. Previous clinical and experimental studies have reported that the reduction of heparin dose with the use of heparin-coated circuits was reduced platelet loss [1, 2] and blood loss [3, 4]. It is considered that there are two effects of the use of heparin-coated equipment and the reduction of heparin dose in this strategy. However, it is not clear whether the beneficial effects on platelets are induced by the use of heparin-coated equipment or by the reduction of heparin dose. We have reported previously that the concomitant use of Duraflo II (Bentley/Baxter, Irvine, CA) heparin-coated equipment with standard systemic heparinization reduces platelet loss and suppresses the platelet release reaction [5]. To clarify the effect of a reduced heparin dose on platelets, we compared standard-dose heparinization and low-dose heparinization.
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Material and methods
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Patients
This prospective study was performed on 23 consecutive patients undergoing elective coronary artery bypass grafting (CABG) using CPB. Patients were stratified according to the number of distal anastomosis (three or fewer versus more than four). They were then randomly assigned to the two groups. An assignment was masked for a patient, an anesthesiologist and surgeons, but was not masked for perfusionists. Eleven patients were assigned to standard-dose systemic heparinization (control group), and 12 patients to low-dose systemic heparinization (low-dose group). The following exclusion criteria were used: (1) preexisting coagulation disorders; (2) ongoing antithrombosis therapy with drugs such as aspirin or warfarin within 1 week before the operation; (3) repeat CABG; (4) severe left ventricular dysfunction (ejection fraction < 0.3); (5) preoperative intraaortic balloon pump; (6) CPB time of less than 90 minutes; and (7) transfusion of more than 3 U of blood during CPB. Informed consent was obtained from all patients.
Bypass circuits and operation
In both groups, all surfaces in potential contact with blood were treated with heparin (Duraflo II). The CPB equipment consisted of a two-stage venous cannula, a venous line, a cardiotomy reservoir (BMR-3500 Gold; Bentley/Baxter), a hollow fiber oxygenator (Univox Gold; Bentley/Baxter), an arterial line, an arterial filter (AF-1040 D; Bentley/Baxter), and a suction tip and line. A cell-saving device was not used for any of the patients.
Five minutes before initiation of CPB, standard-dose systemic heparinization (300 IU/kg porcine mucosa heparin; Novo Nordisk A/S, Denmark) was performed in the control group and low-dose systemic heparinization (200 IU/kg) was used in the low-dose group. Additional heparin was administered during CPB if the activated clotting time (ACT) was below 400 seconds in the control group, and below 300 seconds in the low-dose group. After CPB, protamine was administered in the same dose as the initial heparin dose. Prostaglandin E1, which inhibits surface-induced platelet activation, was not administered during the operation. Hematocrit was maintained at above 18% during CPB. Packed red cells were infused if the hematocrit dropped to below 18%.
Data collection
Arterial blood samples were taken after induction of anesthesia, after heparin administration, 5, 20, 60, and 90 minutes after the initiation of CPB, at the end of CPB, and 10 minutes after protamine administration. The following parameters were measured: ACT, platelet count, platelet factor 4 (PF4), and ß-thromboglobulin (ß-TG) concentrations. ACT was measured by Hemochron (Model 401, International Technidyne, Edison, NJ). Platelet counts were corrected for the hematocrit and standardized to prebypass values. PF4 and ß-TG concentrations were measured by enzyme-linked immunosorbent assay (Diagnostica Stago, Asnières-sur-seine, France) as parameters of platelet activation.
Sampling for electron microscopy
Sample specimens for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were dissected from the arterial filter used in the CPB equipment after blood perfusion. The samples were rinsed with 0.9% NaCl solution, and then fixed with 2.5% glutaraldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2) for 1 hour at 4°C. After rinsing with 7.5% sucrose in the same buffer, the samples were postfixed with 1% OsO4 buffered with 0.1 mol/L phosphate solution (pH 7.2) for 1 hour, and dehydrated with serially graded alcohols. The sample specimens for SEM were further immersed in butylalcohol, and subsequently freeze dried. After platinum ion-coating, they were observed under an electron microscope (S-2300, Hitachi, Japan). The samples for TEM were embedded, and cut into ultrathin sections with an ultramicrotome (Ultracut S, Reichert-Nissei, Japan). After staining with uranyl acetate and lead citrate, they were observed under an electron microscope (H-7100, Hitachi).
As a method of estimating differences in the cellular adhesion on the filter between the two groups, a ten-grade scale was adopted. This was the same method as adopted by Borowiec and associates [6]. It was defined according to the morphologic changes of the cells and the degree of surface coverage by cells and fibrin on SEM: grade 1 denoted no adhesion to the surface and lack of any morphologic changes. In grade 10 the adhesion was extremely advanced with large deposits and no distinguishable cells in a mass.
Statistical analysis
All data are reported as the means ± standard deviations. Differences in preoperative and intraoperative data between the two groups were analyzed using the unpaired Student’s t test. Comparisons between groups, of corrected platelet counts, and ß-TG and PF4 concentrations, were made using the repeated-measures analysis of variance (ANOVA). Differences in the cellular adhesion as determined by SEM were analyzed by the Mann-Whitney U test for intergroup comparisons. A p value of less than 0.05 was considered to indicate a statistically significant difference.
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Results
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Preoperative patient profile and intraoperative details of both groups are shown in Table 1. No significant differences in any of the parameters were observed between the two groups. The 24-hour postoperative blood loss, and the number of autologous blood transfusions during hospitalization are shown in Table 2, with no significant differences noted between the two groups. The postoperative course was uneventful in all patients.
Additional heparin during CPB was not administered to any of the patients. The mean level of highest ACT during CPB was 770 ± 142 seconds in the control group versus 490 ± 106 seconds in the low-dose group (Fig 1). After administration of protamine sulfate, the ACTs returned to prebypass levels in both groups.
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Fig 1. In all patients, additional heparin was not administerd during cardiopulmonary bypass (CPB). The activated clotting times (ACTs) were significantly higher in the control group than in the low-dose group (p < 0.0001; ANOVA).
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The platelet counts were significantly lower (p < 0.01) during CPB in the control group whereas in the low-dose group, they remained near the base line values (Fig 2). After protamine administration, the platelet counts in the control group were 79% ± 16% compared with 96% ± 13% in the low-dose group.
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Fig 2. The platelet (Plt) counts were significantly lower in the control group than in the low-dose group (p < 0.01; ANOVA). (CPB = cardiopulmonary bypass.)
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The ß-TG concentrations increased gradually during CPB (Fig 3). At the end of CPB, the ß-TG concentrations in the control group were 219 ± 56 ng/mL compared with 207 ± 86 ng/mL in the low-dose group. These differences between the two groups were not significant. Similarly, the PF4 concentrations during CPB increased gradually in both groups (Fig 4). At the end of CPB, the PF4 concentrations in the control group were 119 ± 32 ng/mL compared with 122 ± 56 ng/mL in the low-dose group. There were no significant differences between two groups.
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Fig 3. The ß-thromboglobulin (ß-TG) concentrations during cardiopulmonary bypass (CPB) increased gradually in both groups. There were no significant differences between the two groups.
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Fig 4. The platelet facter 4 (PF4) concentrations during cardiopulmonary bypass (CPB) increased gradually in both groups. There were no significant differences between the two groups.
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After blood perfusion, no large clot formation was observed macroscopically on the surface of the arterial filters in the CPB equipment. The SEM findings of the arterial filters obtained from the low-dose group revealed no cells adherent or a few cells adherent on their surfaces (Fig 5), whereas several cellular and fibrous components were found adhering to the surfaces of half of the filter mesh obtained from the control group (Fig 6). The TEM findings revealed that the morphologic characteristics of almost all of these cellular components resembled those of neutrophil leukocytes, including the findings of several-segmented nuclei, many specific granules (300 to 400 nm in diameter), and azurophil granules (600 to 700 nm in diameter) in their cytoplasm (Fig 7). The arterial filters obtained from the low-dose group showed less adhesion than the filters obtained from the control group (Fig 8: p < 0.01).
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Fig 5. Lower magnified scanning electron micrograph of the arterial filter obtained from the low-dose group. (x350 before 5% reduction.)
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Fig 6. Lower magnified scanning electron micrograph of the arterial filter obtained from the control group. (x350 before 5% reduction.)
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Fig 7. Highly magnified scanning electron micrograph of the neutrophil leukocyte on the surface of the arterial filter meshwork (Fm) obtained from the control group. Segmented nucleus (Sn), small specific granules (arrowheads), and azurophil granules (arrows) are observed in the cytoplasm. (x7,500.)
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Fig 8. Cellular adhesion on the arterial filters approximated by scanning electron microscopy views. There were significant differences between two groups (p < 0.01: Mann-Whitney U test).
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Comment
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Systemic heparinization during CPB suppresses activation of the coagulation system and decreases the risk of catastrophic intravascular clotting. However, high-dose heparinization is associated with somewhat greater postoperative blood loss [7]. Heparin is known to induce platelet aggregation [8, 9] and to inhibit platelet function [10]. Chong and associates [11] reported that the degree of aggregation of platelets increased in a dose-dependent manner with increasing concentrations of heparin. In the present study, platelet counts during CPB in the control group were significantly lower than those in the low-dose group. It was suggested that the reduction of heparin dose suppressed the aggregation of platelets.
After protamine administration, platelet counts in both groups decreased. It is reported that infusion of protamine sulfate for neutralization of heparin induces thrombocytopenia [8, 12]. Heparin–protamine complexes activate the classic complement pathway, which causes leukoactivation [13]. The decrease of platelet counts after protamine administration indicated that the heparin–protamine interaction induced platelet aggregation. Thus, it was considered that reduction of both heparin dose and protamine dose contributed to the reduced extent of platelet loss.
On the other hand, there were no significant differences in the platelet granule release reaction between the two groups. This uncoupling of platelet count and platelet granule release reaction has been reported before [1, 2]. Musial and associates [14] reported that a cysteine-rich peptide, which they called "disintegrin," did not prevent the platelet release reaction, but prevented platelet adhesion and aggregation. These phenomena suggested that platelet adhesion and the platelet release reaction occurred by different mechanisms. If the reduction of heparin dose had the same action as "disintegrin," this uncoupling of platelet count and degranulation could be explained.
There are few reports on electron microscopic studies of the effects of reduction of heparin dose along with the use of heparin-coated circuits [1, 6]. Borowiec and associates [6] reported that heparin coating of arterial filters diminished cellular adhesion to the filter surface during CPB. Furthermore, they reported that adherence of blood cells to arterial filters was lower in patients receiving a 50% dose of intravenous heparin than in patients receiving a 25% dose. However, they did not examine the cells adherent on the filters. According to the TEM findings, in the present study, most adherent cells were neutrophils. Korn and associates [2] reported that in an in vitro model of a heparin-coated circuit, release of elastase as a marker of neutrophil activation was lower following low-dose heparinization than following standard-dose heparinization. Berliner and associates [15] suggested that heparin and C5a might have an effect on the aggregation of polymorphonuclear leukocytes. In the present study, concentrations of plasma elastase were not measured. However, the SEM findings suggested that the reduction of heparin dose suppressed activation of leukocytes. The mechanism for the reduced adherence of neutrophils in the low-dose group remains unknown.
The use of a reduced dose of heparin and heparin-coated circuits was reported to suppress complement activation [2, 16] and leukocyte activation [16]. Nevertheless, there are contrary opinions on the practice of reducing the dose of heparin for systemic heparinization because of the increased risk of thrombin generation [1, 17]. Bannan and associates [1] reported that anticoagulation with a reduced heparin dose (one third of the standard dose) with heparin-coated circuits was not adequate at 360 minutes. Prolonged perfusion of low-dose heparin may induce consumptive coagulopathy. In the present study, as the CPB time in all patients was less than 200 minutes (mean, 139 minutes) and the heparin dose used was two thirds of the standard heparin dose (200 IU/kg), anticoagulation was probably adequate. We have previously reported that the use of heparin-coated equipment lessened platelet loss and suppressed the platelet release reaction [5]. However, an additional effect of the reduction of heparin dose on the platelet release reaction was not recognized in the present study. It is debatable whether the beneficial effects of the reduction of heparin dose on platelet function outweigh the risk of thrombin generation.
In summary, the use of a reduced heparin dose and heparin-coated equipment reduced platelet loss, but did not suppress the platelet release reaction. Furthermore, the reduction of heparin dose reduced adherence of leukocyte to the filter surface. Further studies are required to clarify the mechanism of adherence of leukocytes.
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Accepted for publication January 17, 2000.
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Abstract
Introduction
