Platelet Protection by Aprotinin in Cardiomlmonarv BvDass: Electron Microscopic Study I
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Jacob Lavee, MD, Naphtali Savion, PhD, Aram Smolinsky, MD, Daniel A. Goor, MD, and Rephael Mohr, MD Department of Cardiac Surgery and the Maurice and Gabriela Goldschleger Eye Research Institute, The Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
To evaluate the functional integrity of platelets in patients administered the proteinase inhibitor aprotinin during cardiopulmonary bypass, 20 patients undergoing a complicated and prolonged open heart operation were studied. They were randomized to receive either a high dose of aprotinin (total dose, 6 to 7 X lo6 KIU)before and during cardiopulmonary bypass (10 patients) or a placebo (10 patients). Blood samples were collected preoperatively, at the termination of bypass, and 90 minutes thereafter to assess platelet count and aggregation on extracellular matrix, which was studied by scanning electron microscopy. On a scale of 1 to 4, mean preoperative platelet aggregation grades were similar in both groups (3.5 2 0.5). Postoperatively, at the termination of cardiopulmonary bypass and 90 minutes thereafter, all 10 patients treated with aprotinin revealed normal, un-
changed platelet aggregation (grade, 3.5 f 0.51, whereas all placebo-treated patients showed severely disturbed aggregation (grade, 1.4 f 0.5) (p < 0.001). The platelet count was similar in both groups before and after operation (preoperatively, 182 f 75 x 109/L and 146 & 30 X 109/L, and postoperatively, 87 f 13 X 109/L and 80 f 27 x 109/L for the aprotinin and placebo groups, respectively). Total 24-hour postoperative bleeding and blood requirement were significantly lower in the aprotinin group (371 f 84 mL and 2 f 0.7 units, respectively) compared with the placebo group (608 -t 28 mL and 3.4 f 1.3 units, respectively) (p < 0.01). These results demonstrate that improved postoperative hemostatis is directly related to the complete preservation of platelet function achieved by the protective properties of aprotinin. (Ann Thorac Surg 1992;53:477-81)
T
tinin on blood loss, patients with potentially prolonged bypass times were selected for the study.
he impaired hemostatis observed after cardiac operations is mainly due to impaired platelet function [l-51. Administration of the proteinase inhibitor aprotinin improves intraoperative and postoperative platelet function, thus reducing postoperative blood loss and blood requirements [6, 71. In addition to inhibiting plasmin and kallikrein during cardiopulmonary bypass (CPB), aprotinin improves the hemostatic platelet function by preserving adhesive platelet membrane receptors (glycoprotein Ib) [8], thus preventing platelet activation during contact with perfusion systems. In several previously published studies, we [9, 101 demonstrated that CPB causes a significant decrease in the ability of platelets to interact with the subendothelial layer. Using the extracellular matrix (ECM) model and the scanning electron microscope, it was shown in all patients that platelets in blood samples drawn at the end of CPB cannot form mature aggregates similar to those formed before CPB [9]. The purpose of this study is to evaluate with the ECM model the preservative effect of aprotinin on platelet function after CPB. To better evaluate the effect of aproAccepted for publication Aug 30, 1991. Address reprint requests to Dr Goor, Department of Cardiac Surgery, The Chaim Sheba Medical Center, Tel Hashomer 52621, Israel.
0 1992 by The Society of Thoracic Surgeons
Material and Methods After giving informed consent, 20 patients undergoing potentially prolonged CPB procedures were randomized to receive either a high dose of aprotinin before and during CPB (group 1, 10 patients) or a placebo (group 2, 10 patients) (Table 1).The study group (group 1)received a loading dose of 2 x lo6 KIU of aprotinin (Trasylol; Bayer, Leverkusen, Germany) over 20 minutes before sternotomy, followed by continuous infusion of 0.5 x lo6 KIU per hour until skin closure at the end of the operation. An additional 2 x lo6 KIU was added to the priming volume of the oxygenator. Hence, each patient in the study group received a total dose of 6 to 7 x lo6 KIU of aprotinin. Each patient in the control group (group 2) received equivalent volumes of the placebo solution (0.9% saline solution) at the respective times. The mean age of the patients was 62 f 9 years (range, 41 to 78 years), with no difference between the two groups. Preoperative hemoglobin levels were similar in both groups (see Table l), and no patient had been receiving dipyridamole or aspirin-containing drugs during the 4 weeks preceding the operation. For all patients, a Sams pump (Sams Inc, Ann Arbor, MI) and bubble 0003-4975/92/$5.00
478
LAVEE ET AL PLATELET PRESERVATION BY APROTININ
Ann Thorac Surg 1992;53477-81
Table 1. Clinical and Operative Data“
Variable Age (Y) Sex (malelfemale) Hemoglobin level (g1100 mL) Bypass time (min) Aortic cross-clamp time (min) Lowest body temperature (“C) Procedure CABG Redo CABG Valve replacement CABG + valve replacement CABG + aneurysmectomy a
Group 1 (Aprotinin) (n = 10)
Group 2 (Placebo) (n = 10)
58 f 11 911 13.2 f 0.5 185 f 32 63 f 27 25 f 2.6
65 f 7 713 13.3 f 0.4 167 2 36 56 2 35 25 f 4.3
Where applicable, data are shown as the mean 2 the standard deviation.
coated with ECM and agitated (100 rpm) (Orbit Shaker; Lab-Line Instruments, Melrose Park, IL) for 30 minutes at room temperature. At the end of the incubation period, all blood from the culture dish was collected, and the dish was gently flushed several times with phosphate-buffered saline solution.
Preparation for Scanning Electron Microscopic Study The ECM-coated dishes, now containing platelet aggregates, were fixed in 2.5% phosphate-buffered glutaraldehyde (pH 7.2), washed in the same buffer, and postfixed in 2% osmium tetroxide. The third fixation was in a solution of 2% tannic acid-guanidine hydrochloride. The triple-fixed samples were dehydrated in graded alcohol solutions, and thereafter the alcohol was exchanged for Freon 112 by graded Freon solutions (Du Pont Company, Wilmington, DE). The samples were air-dried, goldcoated, and examined by a Jeol 840 scanning electron microscope (Jeol USA Inc, Peabody, MA).
CABG = coronary artery bypass grafting.
Grading Platelet Aggregates on Extracellular Matrix
oxygenator (Bentley Laboratories, Inc, Irvine, CA) were used. The oxygenators were primed with 1,500 mL of Hartmann’s solution and 500 mL of 5% dextrose solution. The two groups were similar with regard to bypass time, aortic cross-clamp time, and lowest body temperature (see Table 1). All patients were rewarmed to 35°C before discontinuation of CPB. Reversal of the effect of heparin sodium by protamine sulfate was monitored by the activated clotting time [ll]. All pump blood was returned to the patient through the aortic cannula or intravenously from infusion bags without hemoconcentration. None of the patients had to be returned to the operating room because of excessive mediastinal bleeding. Blood samples obtained through a flushed arterial catheter were collected from each patient at the following times: preoperatively (immediately after induction of anesthesia and before administration of heparin), at the termination of CPB, and 90 minutes thereafter. Platelet count was measured with a Coulter-Counter-S-Plus I1 (Luton, England).
As a method of quantitating the aggregation of platelets on ECM as seen by the scanning electron microscope, four distinct aggregation grades were defined according to the various platelet morphological types [9]: In grade 1, the platelets were discoid with a lentiform appearance, have a smooth surface, and lack any pseudopodia. The platelets do not adhere to each other, and each platelet can be seen individually. In grade 2, the platelets display the first signs of activation, and they appear with slender dendritelike pseudopodia, still separated from each other. In grade 3, the aggregation process is more advanced. The platelet body spreads, showing multiple dendritic pseudopodia, and the platelets start to cluster. In this immature aggregate, each platelet can still be identified separately. Grade 4 consists of a mature aggregate in which large clumps of platelets are seen and in which individual platelets are difficult to define. In each sample, 40 scanning electron microscopic fields containing platelets were examined at a magnification of 8,000, and each was given an individual grade. The final aggregation grade of each sample was derived by calculating the arithmetic mean of the individual grades. Although aggregates of different grades could be found in any given sample, a dominant grade for each sample could always be identified. To avoid any element of subjectivity in the grading process, the observers assigned grades blindly, that is, they were not aware of the group to which each sample belonged while grading it. The indication for homologous red blood cell transfusion was a hemoglobin level of less than 100 g/L (10 g/100 mL). Platelet transfusion in active bleeders (more than 200 mL/h in the first 2 postoperative hours) was given when the platelet count was lower than 100 x 1 0 9 L Blood loss from the chest tubes, the number of blood units required, and the total number of blood products transfused (red blood cells, plasma, and platelet units) in the initial 24 hours after the patient arrived in the intensive care unit were recorded for evaluation of clinical hemostatis.
Preparation of Dishes Coated With Extracellular Matrix Corneal endothelial cells were grown on 12 well-type tissue culture plates (Nunc GmbH, Roskilde, Denmark) with 5% dextran T-40 (Pharmacia Fine Chemicals, Uppsala, Sweden) added to the growth medium as previously described [12]. Cultures were washed once with phosphate-buffered saline solution and exposed to 0.5% Triton-X 100 solution in phosphate-buffered saline solution (vol/vol), followed by exposure to ammonium hydroxide solution, 0.1 mom, and washing in phosphate-buffered saline solution. Extracellular matrix-coated dishes containing phosphate-buffered saline solution were stored before use at 4°C for up to 3 months.
Platelet Reactivity With Extracellular Matrix Whole blood (0.5 mL in a citrate buffer) from each sample was added to the tissue culture well (16-mm diameter)
LAVEE ET AL PLATELET PRESERVATION BY APROTININ
Ann Thorac Surg 1992;53:477-81
Fig 1. Comparison of platelet aggregation grades P L T Agg Grade) on extracellular matrix in the two groups before operation, immediately after termination of cardiopulmonary bypass (CI'B), and 90 minutes thereafter.
1
Preop
End of CPB Aprotinin
479
90 min after C 'B
0Placebo
Statistical Analysis Results were expressed as the mean f the standard deviation. Paired and nonpaired t tests were used as applicable for statistical analysis.
Results The preoperative platelet count in group 1(aprotinin) (182 f 75 x 109/L) did not differ significantly from that in group 2 (146 f 30 x 109/L).Similarly both groups showed a significantly lower platelet count after CPB (87 ? 13 x 109/L, group 1, and 80 f 27 x 109/L,group 2). The mean preoperative grade of platelet aggregation was 3.5 ? 0.5 in both groups (Fig 1). At the end of CPB, no change in platelet aggregation grade was noted in any of the patients in the aprotinin group (mean grade, 3.5 k 0.5) (Fig 2; see Fig l), whereas in the placebo-treated group, all patients showed a significant decline to grade 1 or 2 (mean grade, 1.4 f 0.5) (p < 0.001) (Fig 3; see Fig 1). This difference between the two groups remained in the measurements made 90 minutes after CPB (see Figs 1-3). Total 24-hour blood loss in group 1 was significantly lower than that in group 2 (Table 2). Patients in the aprotinin-treated group received fewer homologous red
blood cell units and were exposed to a smaller number of homologous blood products than patients in group 2, mainly because of the use of platelet units in 3 group 2 patients as compared with no group 1 patients (see Table 2). The 24-hour postoperative hemoglobin level was 126 2 9 g/L (12.6 ? 0.9 g/100 mL) in group 1 and 124 f 8 g/L (12.4 f 0.8 g/100 mL) in group 2 (p = not significant).
Comment Several recently published studies [6-81 have shown a significant platelet-protective effect of the proteinase inhibitor aprotinin from damage caused by contact with surfaces of the extracorporeal system. The improved platelet function and hemostatis observed were related to the protective effect of aprotinin on platelet membrane glycoprotein Ib, which is the adhesive receptor [8, 131, and glycoprotein IIb/IIIa, which is a membrane fibrinogen receptor related to aggregation [14-161. In this study, the biochemical explanations for better platelet function in patients treated with aprotinin were strongly supported by scanning electron microscopic evidence of better platelet aggregability on the ECM. Cultured endothelial cells, which closely resemble their in
Fig 2 . Scanning electron micrographs from patient who received aprotinin: (a) before operation (grade 4); (b) end of cardiopulmonary bypass (CPB) (grade 4); and (c) 90 minutes after CPB (grade 4). (x 8,000 before 21% reduction.)
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Ann Thorac Surg 1992;53477-81
LAVEE ET AL PLATELET PRESERVATION BY APROTININ
Fig 3 . Scanning electron micrographs from patient who received placebo: (a) before operation (grade 4); (b) end of cardiopulmonay bypass (CPB) (grade 1); and (c) 90 minutes after CPB (grade 1 ) . ( X 8,000 before 21% reduction.)
vivo counterparts, produce an ECM, secreted exclusively beneath the cell layer. This ECM resembles the vascular subendothelial basal lamina in its organization and macromolecular constituents (fibronectin, laminin, collagens type 111, IV, and V, and sulfated proteoglycans) [12, 171. Denudation of the endothelial cell monolayer leaves the underlying ECM intact and firmly attached to the entire surface of the culture dish. The ECM prepared in this way retains its characteristic amorphous structure and biological properties [12, 171. Several studies [18-211 have shown that the ECM produced either by bovine or human endothelial cells can serve as an excellent in vitro model for the study of platelet-subendothelium interaction. Extracorporeal circulation caused a significant decrease in aggregation grade in all patients in the placebo group (mean aggregation grade after CPB was 1.4 compared with 3.5 before CPB). This deleterious effect of CPB did not appear in any of the 10 patients treated with aprotinin (mean aggregation grade after CPB was 3.5). As platelet aggregation on ECM requires both fibrinogen [19]and the von Willebrand factor [22] as ligands for the activated glycoprotein IIb/IIIa, our model is not sensitive enough to differentiate which domain on the glycoprotein IIb/IIIa was protected by aprotinin. The preserved aggregatory capacity of platelets in the aprotinin group of our study demonstrates that at least part, if not all, of the glycoprotein IIb/IIIa was protected. To evaluate the clinical importance of better postoperative hematological hemostasis, patients selected for this study were undergoing operation requiring a potentially prolonged period of CPB. Indeed, although the aprotinin group underwent more complicated procedures than the placebo group (see Table 1) and therefore had a more prolonged CPB time, aprotinin administration signifi-
Table 2 . Postoperative Bleeding and Blood Requirements Variable 24-Hour bleeding (mL) RBC units Total blood products RBC = red blood cells; units.
Group 1 (Aprotinin)
Group 2 (Placebo)
p Value
371 2 84 2 5 0.7 2.3 2 1.0
608 2 28 3.4 2 1.3 6.8 2 5.1
0.05 0.01
0.001
total blood products = RBC + plasma + platelet
cantly reduced both postoperative blood loss and the need for transfusion of homologous red blood cells or other blood products. These results support those of previously published studies in showing that the improved postoperative platelet function after administration of aprotinin is associated with reduced requirements for blood transfusion, thus reducing the risk of exposure to bloodborne viruses. Although the use of high-dose aprotinin increases the cost of operations, this increase is balanced by the reduced use of homologous blood products. In conclusion, we recommend routine use of aprotinin in patients undergoing heart procedures with the potential for increased blood loss, such as redo operations or procedures requiring a prolonged period of CPB. We greatly appreciate the technical expertise and assistance of Jacob Langsam, MSc, of the Electron Microscopy Unit, Life Sciences Department, Bar Ilan University.
References 1. Mohr R, Golan M, Martinowitz U, Rosner F, Goor DA, Ramot B. Effect of cardiac operation on platelets. J Thorac Cardiovasc Surg 1986;92:434-41. 2. Mohr R, Martinowitz U, Golan M, Luski A, Goor DA, Ramot B. Platelet size and mass as an indicator for platelet transfusion after cardiopulmonary bypass. Circulation 1986; 74(Suppl 3):15?-&3. 3. Mohr R, Martinowitz U, Lavee J, Amroch D, Ramot B, Goor DA. The hemostatic effect of transfusing fresh whole blood versus platelet concentrates after cardiac operations. J Thorac Cardiovasc Surg 1988;96:530-4. 4. Harker LA, Malpass TW, Branson HE, Hessel EA 11, Slighter SJ. Mechanism of abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet dysfunction associated with selective alpha granules release. Blood 1980;56:824-34. 5. Edmunds LH Jr, Ellison N, Colman RW, et al. Platelet function during cardiac operation: comparison of membrane and bubble oxygenators. J Thorac Cardiovasc Surg 1982;83: 805-12. 6. Van Oeveren W, Jansen NJG, Bidstrup BP, et al. Effects of aprotinin on hemostatic mechanisms during cardiopulmonary bypass. Ann Thorac Surg 1987;44:640-5. 7. Royston D, Bidstrup BP, Taylor KM, Sapsford RN. Effect of aprotinin on need for blood transfusion after repeat openheart surgery. Lancet 1987;2:1289-91.
Ann Thorac Surg 1992;53:477-81
8. Van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L, Wildevuur CRH. Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:788-97. 9. Lavee J, Martinowitz U,Mohr R, et al. The effect of transfusion of fresh whole blood versus platelet concentrates after cardiac operations. J Thorac Cardiovasc Surg 1989;97204-12. 10. Golan M, Modan M, Lavee J, et al. Transfusion of fresh whole blood stored (4°C) for short period fails to improve platelet aggregation on extracellular matrix and clinical hemostasis after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:354-60. 11. Esposito RA, Culliford AT, Colvin SB, Thomas SJ, Lackner H, Spencer FC. The role of activated clotting time in heparin administration and neutralization for cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983;85:17445. 12. Gospodarowicz D, Vlodavsky I, Savion N. The extracellular matrix. Endocr Rev 1980;1:201-7. 13. Sakariassen KS, Nievelstein PF, Coller BS, Sixma JJ. The role of platelet membrane glycoproteins Ib and 1%-IIIa in platelet adherence to human artery subendothelium. Br J Haematol 1986;63:681-91. 14. Musial J, Niewiarowski S, Hershock D, Morinelli TA, Colman RW, Edmunds LH Jr. Loss of fibrinogen receptors from the platelet surface during simulated extracorporeal circulation. J Lab Clin Med 1985;105:514-22. 15. Wenger RK, Lukasiewicz H, Mikuta BS, Niewiarowski S, Edmunds LH. Loss of platelet fibrinogen receptors during clinical cardiopulmonary bypass. J Thorac Cardiovasc Surg 1989;97:235-9.
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16. Dechavanne M, French M, Pages J, et al. Significant reduction in the binding of a monoclonal antibody (LYP 19) directed against the IIbiIIIa glycoprotein complex to platelets of patients having undergone extracorporeal circulation. Thromb Haemost 1987;57106-9. 17. Gospodarowicz D, Vlodavsky I, Savion N. The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells. J Supramol Struct 1980;13: 339-72. 18. Vlodavsky I, Eldor A, HyAm E, Atzmon R, Fuks Z. Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina. Thromb Res 1982;28 179-91. 19. Eldor A, Vlodavsky I, Martinowitz U, Fuks Z, Coller BS. Platelet interaction with subendothelial extracellular matrix: platelet-fibrinogen interactions are essential for platelet aggregation but not for the matrix-induced release reaction. Blood 1985;65:1477-83. 20. Booyse FM, Quarfoot AJ, Feder A. Culture-produced subendothelium. I. Platelet interaction and properties. Haemostasis 1982;11:49-53. 21. Sakariassen KS, Aarts PAMM, DeGroot PG, Houdjik WPM, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 1983;10252235. 22. OBrien JR. Shear-induced platelet aggregation. Lancet 1990; 1~711-3.
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Jacob Lavee, MD, Naphtali Savion, PhD, Aram Smolinsky, MD, Daniel A. Goor, MD, and Rephael Mohr, MD Department of Cardiac Surgery and the Maurice and Gabriela Goldschleger Eye Research Institute, The Chaim Sheba Medical Center, Tel Hashomer, and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
To evaluate the functional integrity of platelets in patients administered the proteinase inhibitor aprotinin during cardiopulmonary bypass, 20 patients undergoing a complicated and prolonged open heart operation were studied. They were randomized to receive either a high dose of aprotinin (total dose, 6 to 7 X lo6 KIU)before and during cardiopulmonary bypass (10 patients) or a placebo (10 patients). Blood samples were collected preoperatively, at the termination of bypass, and 90 minutes thereafter to assess platelet count and aggregation on extracellular matrix, which was studied by scanning electron microscopy. On a scale of 1 to 4, mean preoperative platelet aggregation grades were similar in both groups (3.5 2 0.5). Postoperatively, at the termination of cardiopulmonary bypass and 90 minutes thereafter, all 10 patients treated with aprotinin revealed normal, un-
changed platelet aggregation (grade, 3.5 f 0.51, whereas all placebo-treated patients showed severely disturbed aggregation (grade, 1.4 f 0.5) (p < 0.001). The platelet count was similar in both groups before and after operation (preoperatively, 182 f 75 x 109/L and 146 & 30 X 109/L, and postoperatively, 87 f 13 X 109/L and 80 f 27 x 109/L for the aprotinin and placebo groups, respectively). Total 24-hour postoperative bleeding and blood requirement were significantly lower in the aprotinin group (371 f 84 mL and 2 f 0.7 units, respectively) compared with the placebo group (608 -t 28 mL and 3.4 f 1.3 units, respectively) (p < 0.01). These results demonstrate that improved postoperative hemostatis is directly related to the complete preservation of platelet function achieved by the protective properties of aprotinin. (Ann Thorac Surg 1992;53:477-81)
T
tinin on blood loss, patients with potentially prolonged bypass times were selected for the study.
he impaired hemostatis observed after cardiac operations is mainly due to impaired platelet function [l-51. Administration of the proteinase inhibitor aprotinin improves intraoperative and postoperative platelet function, thus reducing postoperative blood loss and blood requirements [6, 71. In addition to inhibiting plasmin and kallikrein during cardiopulmonary bypass (CPB), aprotinin improves the hemostatic platelet function by preserving adhesive platelet membrane receptors (glycoprotein Ib) [8], thus preventing platelet activation during contact with perfusion systems. In several previously published studies, we [9, 101 demonstrated that CPB causes a significant decrease in the ability of platelets to interact with the subendothelial layer. Using the extracellular matrix (ECM) model and the scanning electron microscope, it was shown in all patients that platelets in blood samples drawn at the end of CPB cannot form mature aggregates similar to those formed before CPB [9]. The purpose of this study is to evaluate with the ECM model the preservative effect of aprotinin on platelet function after CPB. To better evaluate the effect of aproAccepted for publication Aug 30, 1991. Address reprint requests to Dr Goor, Department of Cardiac Surgery, The Chaim Sheba Medical Center, Tel Hashomer 52621, Israel.
0 1992 by The Society of Thoracic Surgeons
Material and Methods After giving informed consent, 20 patients undergoing potentially prolonged CPB procedures were randomized to receive either a high dose of aprotinin before and during CPB (group 1, 10 patients) or a placebo (group 2, 10 patients) (Table 1).The study group (group 1)received a loading dose of 2 x lo6 KIU of aprotinin (Trasylol; Bayer, Leverkusen, Germany) over 20 minutes before sternotomy, followed by continuous infusion of 0.5 x lo6 KIU per hour until skin closure at the end of the operation. An additional 2 x lo6 KIU was added to the priming volume of the oxygenator. Hence, each patient in the study group received a total dose of 6 to 7 x lo6 KIU of aprotinin. Each patient in the control group (group 2) received equivalent volumes of the placebo solution (0.9% saline solution) at the respective times. The mean age of the patients was 62 f 9 years (range, 41 to 78 years), with no difference between the two groups. Preoperative hemoglobin levels were similar in both groups (see Table l), and no patient had been receiving dipyridamole or aspirin-containing drugs during the 4 weeks preceding the operation. For all patients, a Sams pump (Sams Inc, Ann Arbor, MI) and bubble 0003-4975/92/$5.00
478
LAVEE ET AL PLATELET PRESERVATION BY APROTININ
Ann Thorac Surg 1992;53477-81
Table 1. Clinical and Operative Data“
Variable Age (Y) Sex (malelfemale) Hemoglobin level (g1100 mL) Bypass time (min) Aortic cross-clamp time (min) Lowest body temperature (“C) Procedure CABG Redo CABG Valve replacement CABG + valve replacement CABG + aneurysmectomy a
Group 1 (Aprotinin) (n = 10)
Group 2 (Placebo) (n = 10)
58 f 11 911 13.2 f 0.5 185 f 32 63 f 27 25 f 2.6
65 f 7 713 13.3 f 0.4 167 2 36 56 2 35 25 f 4.3
Where applicable, data are shown as the mean 2 the standard deviation.
coated with ECM and agitated (100 rpm) (Orbit Shaker; Lab-Line Instruments, Melrose Park, IL) for 30 minutes at room temperature. At the end of the incubation period, all blood from the culture dish was collected, and the dish was gently flushed several times with phosphate-buffered saline solution.
Preparation for Scanning Electron Microscopic Study The ECM-coated dishes, now containing platelet aggregates, were fixed in 2.5% phosphate-buffered glutaraldehyde (pH 7.2), washed in the same buffer, and postfixed in 2% osmium tetroxide. The third fixation was in a solution of 2% tannic acid-guanidine hydrochloride. The triple-fixed samples were dehydrated in graded alcohol solutions, and thereafter the alcohol was exchanged for Freon 112 by graded Freon solutions (Du Pont Company, Wilmington, DE). The samples were air-dried, goldcoated, and examined by a Jeol 840 scanning electron microscope (Jeol USA Inc, Peabody, MA).
CABG = coronary artery bypass grafting.
Grading Platelet Aggregates on Extracellular Matrix
oxygenator (Bentley Laboratories, Inc, Irvine, CA) were used. The oxygenators were primed with 1,500 mL of Hartmann’s solution and 500 mL of 5% dextrose solution. The two groups were similar with regard to bypass time, aortic cross-clamp time, and lowest body temperature (see Table 1). All patients were rewarmed to 35°C before discontinuation of CPB. Reversal of the effect of heparin sodium by protamine sulfate was monitored by the activated clotting time [ll]. All pump blood was returned to the patient through the aortic cannula or intravenously from infusion bags without hemoconcentration. None of the patients had to be returned to the operating room because of excessive mediastinal bleeding. Blood samples obtained through a flushed arterial catheter were collected from each patient at the following times: preoperatively (immediately after induction of anesthesia and before administration of heparin), at the termination of CPB, and 90 minutes thereafter. Platelet count was measured with a Coulter-Counter-S-Plus I1 (Luton, England).
As a method of quantitating the aggregation of platelets on ECM as seen by the scanning electron microscope, four distinct aggregation grades were defined according to the various platelet morphological types [9]: In grade 1, the platelets were discoid with a lentiform appearance, have a smooth surface, and lack any pseudopodia. The platelets do not adhere to each other, and each platelet can be seen individually. In grade 2, the platelets display the first signs of activation, and they appear with slender dendritelike pseudopodia, still separated from each other. In grade 3, the aggregation process is more advanced. The platelet body spreads, showing multiple dendritic pseudopodia, and the platelets start to cluster. In this immature aggregate, each platelet can still be identified separately. Grade 4 consists of a mature aggregate in which large clumps of platelets are seen and in which individual platelets are difficult to define. In each sample, 40 scanning electron microscopic fields containing platelets were examined at a magnification of 8,000, and each was given an individual grade. The final aggregation grade of each sample was derived by calculating the arithmetic mean of the individual grades. Although aggregates of different grades could be found in any given sample, a dominant grade for each sample could always be identified. To avoid any element of subjectivity in the grading process, the observers assigned grades blindly, that is, they were not aware of the group to which each sample belonged while grading it. The indication for homologous red blood cell transfusion was a hemoglobin level of less than 100 g/L (10 g/100 mL). Platelet transfusion in active bleeders (more than 200 mL/h in the first 2 postoperative hours) was given when the platelet count was lower than 100 x 1 0 9 L Blood loss from the chest tubes, the number of blood units required, and the total number of blood products transfused (red blood cells, plasma, and platelet units) in the initial 24 hours after the patient arrived in the intensive care unit were recorded for evaluation of clinical hemostatis.
Preparation of Dishes Coated With Extracellular Matrix Corneal endothelial cells were grown on 12 well-type tissue culture plates (Nunc GmbH, Roskilde, Denmark) with 5% dextran T-40 (Pharmacia Fine Chemicals, Uppsala, Sweden) added to the growth medium as previously described [12]. Cultures were washed once with phosphate-buffered saline solution and exposed to 0.5% Triton-X 100 solution in phosphate-buffered saline solution (vol/vol), followed by exposure to ammonium hydroxide solution, 0.1 mom, and washing in phosphate-buffered saline solution. Extracellular matrix-coated dishes containing phosphate-buffered saline solution were stored before use at 4°C for up to 3 months.
Platelet Reactivity With Extracellular Matrix Whole blood (0.5 mL in a citrate buffer) from each sample was added to the tissue culture well (16-mm diameter)
LAVEE ET AL PLATELET PRESERVATION BY APROTININ
Ann Thorac Surg 1992;53:477-81
Fig 1. Comparison of platelet aggregation grades P L T Agg Grade) on extracellular matrix in the two groups before operation, immediately after termination of cardiopulmonary bypass (CI'B), and 90 minutes thereafter.
1
Preop
End of CPB Aprotinin
479
90 min after C 'B
0Placebo
Statistical Analysis Results were expressed as the mean f the standard deviation. Paired and nonpaired t tests were used as applicable for statistical analysis.
Results The preoperative platelet count in group 1(aprotinin) (182 f 75 x 109/L) did not differ significantly from that in group 2 (146 f 30 x 109/L).Similarly both groups showed a significantly lower platelet count after CPB (87 ? 13 x 109/L, group 1, and 80 f 27 x 109/L,group 2). The mean preoperative grade of platelet aggregation was 3.5 ? 0.5 in both groups (Fig 1). At the end of CPB, no change in platelet aggregation grade was noted in any of the patients in the aprotinin group (mean grade, 3.5 k 0.5) (Fig 2; see Fig l), whereas in the placebo-treated group, all patients showed a significant decline to grade 1 or 2 (mean grade, 1.4 f 0.5) (p < 0.001) (Fig 3; see Fig 1). This difference between the two groups remained in the measurements made 90 minutes after CPB (see Figs 1-3). Total 24-hour blood loss in group 1 was significantly lower than that in group 2 (Table 2). Patients in the aprotinin-treated group received fewer homologous red
blood cell units and were exposed to a smaller number of homologous blood products than patients in group 2, mainly because of the use of platelet units in 3 group 2 patients as compared with no group 1 patients (see Table 2). The 24-hour postoperative hemoglobin level was 126 2 9 g/L (12.6 ? 0.9 g/100 mL) in group 1 and 124 f 8 g/L (12.4 f 0.8 g/100 mL) in group 2 (p = not significant).
Comment Several recently published studies [6-81 have shown a significant platelet-protective effect of the proteinase inhibitor aprotinin from damage caused by contact with surfaces of the extracorporeal system. The improved platelet function and hemostatis observed were related to the protective effect of aprotinin on platelet membrane glycoprotein Ib, which is the adhesive receptor [8, 131, and glycoprotein IIb/IIIa, which is a membrane fibrinogen receptor related to aggregation [14-161. In this study, the biochemical explanations for better platelet function in patients treated with aprotinin were strongly supported by scanning electron microscopic evidence of better platelet aggregability on the ECM. Cultured endothelial cells, which closely resemble their in
Fig 2 . Scanning electron micrographs from patient who received aprotinin: (a) before operation (grade 4); (b) end of cardiopulmonary bypass (CPB) (grade 4); and (c) 90 minutes after CPB (grade 4). (x 8,000 before 21% reduction.)
480
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LAVEE ET AL PLATELET PRESERVATION BY APROTININ
Fig 3 . Scanning electron micrographs from patient who received placebo: (a) before operation (grade 4); (b) end of cardiopulmonay bypass (CPB) (grade 1); and (c) 90 minutes after CPB (grade 1 ) . ( X 8,000 before 21% reduction.)
vivo counterparts, produce an ECM, secreted exclusively beneath the cell layer. This ECM resembles the vascular subendothelial basal lamina in its organization and macromolecular constituents (fibronectin, laminin, collagens type 111, IV, and V, and sulfated proteoglycans) [12, 171. Denudation of the endothelial cell monolayer leaves the underlying ECM intact and firmly attached to the entire surface of the culture dish. The ECM prepared in this way retains its characteristic amorphous structure and biological properties [12, 171. Several studies [18-211 have shown that the ECM produced either by bovine or human endothelial cells can serve as an excellent in vitro model for the study of platelet-subendothelium interaction. Extracorporeal circulation caused a significant decrease in aggregation grade in all patients in the placebo group (mean aggregation grade after CPB was 1.4 compared with 3.5 before CPB). This deleterious effect of CPB did not appear in any of the 10 patients treated with aprotinin (mean aggregation grade after CPB was 3.5). As platelet aggregation on ECM requires both fibrinogen [19]and the von Willebrand factor [22] as ligands for the activated glycoprotein IIb/IIIa, our model is not sensitive enough to differentiate which domain on the glycoprotein IIb/IIIa was protected by aprotinin. The preserved aggregatory capacity of platelets in the aprotinin group of our study demonstrates that at least part, if not all, of the glycoprotein IIb/IIIa was protected. To evaluate the clinical importance of better postoperative hematological hemostasis, patients selected for this study were undergoing operation requiring a potentially prolonged period of CPB. Indeed, although the aprotinin group underwent more complicated procedures than the placebo group (see Table 1) and therefore had a more prolonged CPB time, aprotinin administration signifi-
Table 2 . Postoperative Bleeding and Blood Requirements Variable 24-Hour bleeding (mL) RBC units Total blood products RBC = red blood cells; units.
Group 1 (Aprotinin)
Group 2 (Placebo)
p Value
371 2 84 2 5 0.7 2.3 2 1.0
608 2 28 3.4 2 1.3 6.8 2 5.1
0.05 0.01
0.001
total blood products = RBC + plasma + platelet
cantly reduced both postoperative blood loss and the need for transfusion of homologous red blood cells or other blood products. These results support those of previously published studies in showing that the improved postoperative platelet function after administration of aprotinin is associated with reduced requirements for blood transfusion, thus reducing the risk of exposure to bloodborne viruses. Although the use of high-dose aprotinin increases the cost of operations, this increase is balanced by the reduced use of homologous blood products. In conclusion, we recommend routine use of aprotinin in patients undergoing heart procedures with the potential for increased blood loss, such as redo operations or procedures requiring a prolonged period of CPB. We greatly appreciate the technical expertise and assistance of Jacob Langsam, MSc, of the Electron Microscopy Unit, Life Sciences Department, Bar Ilan University.
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