- Email: [email protected]
Thrombomodulin in pediatric cardiac surgery
Thrombomodulin in Pediatric Cardiac Surgery Joachim Boldt, MD, Christoph Knothe, MD, Ehrenfried Schindler, MD, Annegret Welters, Friedhelm F. Dapper, MD, and Gunter Hempelmann, MD Department of Anesthesiology and Intensive Care Medicine and Department of Cardiovascular Surgery, Justus-Liebig-University Giessen, Giessen, Germany
In 30 consecutive children with congenital heart disease scheduled for pediatric cardiac operations, thrombomodd i n , protein C, free protein S, and thrombin-antithrombin complex were measured by enzyme-linked immunosorbent assay after the induction of anesthesia (baseline value), and then before, during, and after cardiopulmonary bypass until the first postoperative day. The patients were divided prospectively into two groups: children weighing less than 10 kg (group l; n = 15)and those weighing more than 10 kg (group 2; n = 15). At baseline, the plasma concentration of thrombomodulin was significantly higher in the children in group 1 than in those in group 2 (83.1 & 11.0 ng/mL versus 29.2 & 12.1 ng/mL). During cardiopulmonary bypass, the thrombomodulin level was reduced in both groups without showing any significant group differences. Five hours after cardiopulmonary bypass and on the first postoperative day, the thrombomodulin level exceeded normal values only in the children weighing less than 10 kg. In both groups, the protein C levels were already below normal at the beginning of the study. The baseline protein S concentration was higher in the smaller children (80% & 18%) than in the larger children (66% & 11%). It was reduced
C
ardiopulmonary bypass (CPB) is known to profoundly influence plasma- and platelet-associated coagulation [l].This may result either in prolonged or enhanced bleeding, or both, or in early thrombosis stemming from contact activation by the nonendothelial synthetic surfaces of the extracorporeal circulation equipment. In recent years, it has become obvious that the vascular endothelium is metabolically active, and this includes the regulation of pressure and hemostasis [2, 31. The vascular endothelial cells produce various important antithrombotic substances such as plasmin activators, heparinlike enzymes, and others [3]. Thrombomodulin (TM) is one of these endothelial cell products that contributes to the regulation of coagulation [4-6]. A growing body of data indicates that TM plays an important role in maintaining the anticoagulant surface of the endothelium [6].Expression of TM from the endothelial surface is activated by various stimuli, including low blood flow and hypoxia [7]. Accepted for publication Oct 20, 1993. Address reprint requests to Dr Boldt, Department of Anesthesiology and Intensive Care Medicine, Justus-Liebig-University Giessen, Klinikstrasse 29, 35392 Giessen, Germany.
0 1994 by The Society of Thoracic Surgeons
by cardiopulmonary bypass in both groups; however, postoperatively it did not return to normal in group 1 (45.1% 2 10%). Plasma levels of the thrombinantithrombin complex were similar in both groups, with a marked increase at the end of cardiopulmonary bypass, and returned to near-normal levels by 5 hours after bypass. Other coagulation variables (the partial thromboplastin time and the antithrombin 111 and fibrinogen levels) were not significantly different between the two groups. Postoperative blood loss was significantly higher in the smaller children on the first postoperative day (35.3 & 20.1 mL/kg) than in the children weighing more than 10 kg (11.8 2 5.0 mL/kg). Five children in group 1 and 2 children in group 2 received fresh frozen plasma in the postbypass period (p < 0.05). It is concluded that the endothelium is involved in the regulation of hemostasis by producing the natural anticoagulant thrombomodulin. This endothelium-related system was significantly more altered in the smaller children with congenital heart disease undergoing cardiac operations, than it was in the bigger children, which may have contributed to the greater bleeding tendency in these children. (Ann Thorac Surg 1994;57:1584-9)
Thrombin binds to TM, and then this thrombin-TM complex activates protein C, which is known to be a potent anticoagulant. Together with protein S as a cofactor, activated protein C degrades coagulation factors Va and VIIIa, thereby limiting clotting activity [3, 51. Thus, abnormalities in TM production may result in abnormal regulation of coagulation [6]. Only very little is known of this complex interplay between endothelium and coagulation in patients suffering from congenital heart disease. Thus, the present study was carried out to investigate changes in the plasma levels of TM in pediatric patients undergoing cardiac operations using CPB.
Material and Methods Pa tients Thirty consecutive children scheduled for palliative or corrective operations for the treatment of congenital heart disease were included in this prospectively designed study. Informed consent was obtained from the parents according to the protocol of the ethics study committee of the hospital. Exclusion criteria were a reoperation, use of anticoagulant drugs within 10 days before the operation (heparin, cyclooxygenase inhibitors), and renal insuffi0003-4975/94/$7.00
Ann Thorac Surg 1994;57:1584-9
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
ciency (creatinine level exceeding 1.5 mg/dL). The children were divided into two groups according to weight. Group 1 consisted of 15 children weighing less than 10 kg; group 2 was made up of 15 children weighing between 10 and 25 kg. The anesthesia regimen was similar for all patients, and consisted of weight-related doses of fentanyl (total dose, 0.05 ? 0.02 mg/kg), midazolam (total dose, 0.65 2 0.03 mg/kg), and pancuronium bromide (total dose, 0.35 k 0.05 mg/kg). All patients were ventilated mechanically at least until the first postoperative day. Ventilation patterns were selected according to pulse oximetry, endexpiratory carbon dioxide, and blood gas analysis values. No volatile anesthetics were used during the investigation period. Anesthesia was always administered by the same anesthesiologist, who was not involved in the study.
Measured Variables and Data Points
Cardiopulmonary Bypass Five minutes before the start of CPB, 300 U/kg of bovine heparin preparation was administered to achieve anticoagulation. The activated clotting time was kept at greater than 400 seconds during the entire bypass period. A COBE VPCMLplus membrane oxygenator (Cobe Laboratories, Lakewood, CO) was used, and a flow of 2.4 L/min * m2 was maintained during CPB. Priming of the extracorporeal circuit consisted of Ringer's solution, 5% human albumin, and electrolytes. One unit of packed red blood cells was added to the prime in all children weighing less than 10 kg. In the group of children weighing more than 10 kg, packed red blood cells were added with regard to the preoperative hematocrit value (packed red blood cells were added when the preoperative hematocrit was less than 0.35). The amount of the priming volume was identical in all children (800 mL). Additional packed red blood cells were given when the hematocrit was less than 0.20. When necessary, Ringer's solution was added to maintain the filling of the circuit. After the end of CPB, the blood remaining in the extracorporeal oxygenation equipment was salvaged by a centrifugation device (Cell Saver 111; Haemonetics, Braintree, MA). All autologous blood was retransfused in the postbypass period. Heparin was neutralized by the administration of protamine chloride in a ratio of 1:l with regard to the initially given dose of heparin. Further protamine was given when the activated clotting time exceeded 200 seconds. All children were operated on by the same surgeon. The decision to administer packed red blood cells and blood derivatives (fresh frozen plasma, platelet concentrates) was made by physicians who were not involved in the study. Packed red blood cells were given when the hematocrit was less than 0.30. After careful exclusion of surgical bleeding, administration of fresh frozen plasma was indicated postoperatively when bleeding exceeded 5 mL * kg-' * h-' and the platelet count exceeded 50,00O/mL (activated clotting time, <200 seconds). Platelet concentrates were administered when bleeding exceeded 5 mL * kg-' h-' and the platelet count was less than 50,00O/mL (activated clotting time, (200 seconds).
1585
Hemoglobin, hematocrit, and standard coagulation variables (activated clotting time, antithrombin 111, and fibrinogen plasma concentrations, as well as the partial thromboplastin time were determined in arterial blood samples. The TM plasma levels were measured by a one-step sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody (Diagnostica Stago, Asnieres Cedex, France) raised against human TM [8]. The ELISA used two monoclonal antibodies that react at different sites of the TM molecule and do not interfere with each other's binding. Protein C and S plasma concentrations were also assessed by ELISA (Boehringer-Mannheim, Mannheim, Germany). Protein S was quantified as free protein S after bound protein S was removed from the plasma by precipitation with polyethylene (normal for free protein S, >30%). Normal values of TM in healthy volunteers determined with this method were reported to be less than 40 ng/mL [lo]; the protein C concentration normally ranged from 70% to 110%. The thrombinantithrombin complex was also measured by ELISA (Behringwerke, Marburg, Germany [normal values, <30-40 pg/L]). All data from the ELISAs represent the means of duplicate measurements. Measurements were performed after induction of anesthesia (the baseline values), before the start of CPB (before anticoagulation with heparin), 20 minutes after the onset of CPB, immediately after the end of CPB (before infusion of protamine), at the end of operation, 5 hours after the end of CPB, and on the morning of the first postoperative day. The quantities of fluids administered (including fresh frozen plasma and packed red blood cells) were recorded and the urine output, blood loss from postbypass suction, and chest tube drainage were also documented.
Stat is t ics Mean values and standard deviations were calculated for all variables. Statistical interpretation was performed by multivariate (repeated-measures) analysis of variance (including multiple-range tests [Scheffcs test]). 2 analysis was performed for differences in homologous blood use. Values of p less than 0.05 were considered significant.
Results The patients' characteristics and the data from CPB are shown in Table 1.The children in group 1 ( < l o kg) ranged in age from 3 days to 13 months and their body weight ranged from 3.7 to 7.9 kg. The children in group 2 (>lo kg) ranged in age from 35 to 68 months and their weight ranged from 13.3 to 22.5 kg. There were no differences among the groups with regard to the duration of the bypass period, the lowest temperatures during bypass, and the number of cyanotic and acyanotic congenital heart diseases. The breakdown of the surgical procedures was similar for both groups (Table 2). The degree of hemodilution (hemoglobin) and the values for standard coagulation variables (antithrombin 111, fibrinogen, partial thromboplastin time, platelet count)
1586
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
Table 1 . Patients’ Characteristics and Data From Cardiopulmonaru Bupass Patient Characteristics Age (mo) Weight (kg) Cyanotic Acyanotic CPB (min) Ischemia”(min) Total heparin (units) Lowest temperature (“C) Rectal Nasopharyngeal Tube drainage (mLkg) 5 hr after CPB 1st postoperative day Use of homologous blood and products (except PRC added to the prime) (mL) PRC FFP a
Period of aortic cross-clamping.
CPB = cardiopulmonary bypass; = packed red blood cells.
Group 1 (
Group 2 (1 ’0
kg)
7.0 f 3.3 5.5 f 1.0 6 9 157 f 55 95 f 32 2,677 f 641
54.2 ? 10.lb 16.1 ? 2.0b 7 8 154 f 42 87 ? 45 10,532 ? 1,59Bb
30.2 f 4.1 28.3 2 4.4
31.7 ? 2.6 29.0 f 3.7
11.6 2 6.9 35.3 f 20.1
3.4 2 2.1b 11.8 2 5.0b
610 285
230b 150
p < 0.05. FFP = fresh frozen plasma;
PRC
did not differ significantly between the two groups (Table 3). Five hours after CPB and on the first postoperative day, the postoperative blood loss was significantly higher in group 1 than in group 2 (see Table 1). The total use of homologous blood and blood products in the postoperative period was greater in the smaller children (Table 1):6 and 5 children in group 1required packed red blood cells and fresh frozen plasma, respectively, compared to 2 and 2 children, respectively, in group 2 ( p < 0.05). The TM plasma concentration at baseline was significantly higher in group 1 than in group 2 children (Fig 1). The TM concentration was not influenced by the anesthesia or the surgical procedure itself (no change from the level after induction until the level before the start of CPB). During CPB, the TM concentration was reduced in both groups without showing any significant differences between the groups. Five hours after CPB and on the first postoperative day, the TM level exceeded normal values in the group 1 children, but it almost returned to normal in the group 2 children. The protein C level was already below normal in both groups at the beginning of the study (approximately59%) (Fig 2). During CPB and immediately after, the decrease was more pronounced in group 1 children (from 59% to 19%) than in group 2 children (from 59% to 39%). Postoperatively, it remained below normal in both groups without any significant differences between the groups. The protein S level was higher at baseline in the smaller children (80%), although it was normal in the children weighing more than 10 kg (66%) (see Fig 2). In children
Ann Thorac Surg 1994;571584-9
weighing less than 10 kg (group l), the protein S plasma level did not return to the baseline level postoperatively (45.1% -C 10%). The thrombin-TM complex values showed a similar course in both groups, with a marked increase at the end of CPB and at the end of the operation (Fig 3). The values had almost normalized by 5 hours after the end of CPB in both groups.
Comment Coagulation abnormalities that occur in children undergoing pediatric cardiac operations represent a complex interrelationship between preexisting coagulation defects and coagulation defects secondary to CPB [9]. Wyss and associates [lo] reported that 11 of the 20 children in their study who underwent cardiac operations exhibited various coagulation problems preoperatively. After CPB, additional alterations in the coagulation status were observed. In addition to platelet-related and factor-associated coagulation abnormalities, endothelium-derived changes constitute a new dimension as a source of disturbances in coagulation. Thrombomodulin is a surface protein expressed on the endothelium that is involved in the regulation of coagulation. It neutralizes thrombin clotting activity and accelerates the thrombin-catalyzed activation of protein C [3, 5, 111. Thus, together with protein C, it represents an important physiologic anticoagulant system 13, 121. One major finding in the present study was that, after the induction of anesthesia (before CPB), the TM level was significantly higher in the children weighing less than 10 kg than in the children weighing more than 10 kg. During and immediately after CPB, the TM concentration decreased significantly in both groups-most likely due to hemodilution during CPB. Postoperatively, the TM concentration increased beyond normal values only in the group 1 children. Normal TM plasma levels (in healthy adults) are not clearly defined. It appears to be highly dependent on the ELISA method used for measuring the TM concentration. With the method used in the present study, the normal values of TM were found to be 30 to 40 pg/L [13]. Using different assays, other authors have
Table 2. Type of Operation in the Two Groups Type of Operation Fallot IV repair TGA ASD + VSD repair PST + ASD repair PST + VSD repair Aortopulmonary shunt Mitral valve replacement AV canal repair Homograft
Group 1 (
Group 2 (’10 kg)
2 2 1 1 1 2
1 1 2
... 3 2
...
2
2 2
2 2
IV = intravenAV = atrioventricular; ASD = atrial septal defect; PST = stenosis of the pulmonary artery; TGA = transposition tricular; VSD = ventricular septal defect. of the great arteries;
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
Ann Thorac Surg 1994;57:15849
1587
Table 3. Hemoglobin, Antithrombin III, and Fibrinogen Plasma Levels and the Partial Thromboplastin Time in the Two Groups Hematologic Variable
After Induction
Before CPB
Hemoglobin (g/dL) <10 kg >10 kg Antithrombin 111 (%)”
14.0 f 1.1 12.7 t 2.1
13.4 & 0.9 12.2 f 2.2
77 +. 12 80 2 11
77 & 10 79 t 10
199 2 22 220 2 24
195 t 20 205 f 21
>10 kg Fibrinogen (g/L)b 4 0 kg >10 kg PTT (s)c 4 0 kg >10 kg Normal values: a 70%-110%; CBP
=
50 52
50 f 11 54 2 10 1 . 5 4 0 g/L;
cardiopulmonary bypass;
p.0. =
f f
After CPB
End of Operation
5 Hours after CPB
1st p.0. Day
10.4 f 1.1 10.1 t 1.9
10.3 f 0.7 11.9 f 1.4
12.3 t 1.8 12.6 +- 2.2
13.7 f 1.9 12.5 t 1.8
50 f 11 56 t 11
53 5 8 49 f 10
49 2 8 56 & 12
8 9 f 11 90 f 13
98 2 12 107 & 14
109 & 22 125 f 29
>300 >300
>300 >300
During CPB 8.3 9.2
11 9
f &
0.7 0.9
10 9
58 t 11 60 2 9
142 t 22 139 & 28
253 t 32 287 f 33
12 11
58 & 11 48 2 14
60 59
64 f 11 51 ? 12
56 46
f f
f f
25-35 seconds.
PTT = partial thromboplastic time
postoperative;
separated children according to weight because the child’s weight appears to be more closely related to maturity than the child’s age. Increased levels of TM may either result from (generalized) endothelial alterations or kidney dysfunction [3, 161. As renal insufficiency was ruled out by our exclusion
6o
50 40
-
-
30
I (ng/ml 110 100
I00
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
--
*
:
----
protein S
(%)
1
10 0
I
I
after induction
I
before CPB
I
CPB
I
after CPB
I
I
I
end of 5 hrs 1. p.0 operation after CPB day
Fig 1. Thrombomodulin plasma levels (in nanograms per milliliter) in the two groups (normal values in the adult, < 3 U O nglmL). (CPB = cardiopulmonary bypass; p.0. = postoperative; SD = standard deviation.)
after before induction CPB
CPB
after CPB
end of
5 hrs
operation after CPB
1 . p.0 day
Fig 2. Protein C and (free) protein S plasma concentrations (normal values in the adult: protein C, 70% to 110%;free protein S, >30%). (CPB = cardiopulmonary bypass; p.0. = postoperative; SD = standard deviation.)
1588
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
240 220 200 I80
160 I40 I20 I00 80 60 40
20
after induction
I
I
I
I
before CPB
CPB
after CPB
-B-
I
I
I
end of 5 hrs I . p.0 operation after CPB day
-0-
>IOkg
I
Fig 3 . Thrombin-antithrombin plasma levels in the two groups (normal values in the adult, <30 pgFL). (CPB = cardiopulmonary bypass; p.0. = postoperative; SD = standard deviation.)
to endothelial cell damage [13]. Hypoxemia may cause alterations in plasma- and platelet-associated coagulation [17]. Hypoxia, however, was reported to lead to a decrease in TM activity on the cell surface [7]. Moreover, there were no differences among the two groups in the present study in terms of the number of cyanotic and acyanotic congenital heart diseases. In the setting of disseminated intravascular coagulation and pulmonary insufficiency, the soluble TM level was reported to be significantly increased [3, 181. In the disseminated intravascular coagulation syndrome, activated proteolytic enzymes in the coagulation-fibrinolysis system may induce release of TM. In the setting of the adult respiratory distress syndrome, proteases released from leukocytes may be responsible for the release of TM into the circulation [4]. The thrombin-antithrombin complex plasma levels (and standard coagulation variables) at the beginning of the study showed no evidence of activation of the coagulation system leading to thrombin generation [ 19, 201. As known for other endothelium-derived substances (eg, endothelin, endothelium-derived releasing factor), the maintenance of blood fluidity and microcirculatory blood flow may also play a central role in maintaining endothelial integrity [6], by which TM expression and release may be influenced. It is difficult to decide whether the smaller (younger) children in the present study were more ill, and thus affected by more altered hemodynamics, than were the children weighing more than 10 kg. It also cannot be definitely concluded from the present results just how far changes in the circulating TM levels represent changes in endothelial expression, and vice versa. One problem with endothelium-associated substances is that they are expressed on the endothelial surface. Although TM is mainly found on endothelial cell surfaces, it is also present in circulating blood [21]. Thrombomodulin also appears to be active in its soluble form: when TM
Ann Thorac Surg 1994;5715%9
was purified from plasma, it was found to catalyze protein C activation by thrombin. The apparent Michaelis constant for protein C was identical for both the soluble and cellular forms of TM. The protein C concentration was most markedly reduced by CPB in the small children. This may be due to an increased activation and increased elimination by the reticuloendothelial system [22]. Lowered plasma levels of protein C have been observed during in vivo activation of the blood coagulation system [23]. In this situation, this may be due to the clearance of protein C activated by thrombin [24]. The protein C level remained lower in the children weighing less than 10 kg than in those weighing more than 10 kg until the end of the investigation period. In adults undergoing cardiac procedures, Knob1 and associates [20] also found a decreased protein C plasma concentration during and after CPB. They assumed that this might be one reason for the well-known bleeding tendency seen in patients undergoing cardiac procedures. One explanation for the reduced protein C levels in the small children ( < l o kg) in the present study might be a failure to adequately synthesize the quantities necessary to replenish the protein C losses that occur during CPB. Bleeding was greater in our group 1 children and the altered TM-protein C system may additionally contribute to this phenomenon. The baseline protein S plasma levels were also significantly lower in the smaller children, and were still reduced in the postoperative period. This agrees with observations made in adults, who showed a significant decrease in the TM, protein C, and protein S plasma concentrations during CPB [20]. This decrease is considered to reflect the activation and consumption of the TM-protein C system in response to thrombin generation. The decrease in the present study, however, was much more pronounced than that seen in adult patients undergoing cardiac procedures. It is concluded from our findings that the imbalance in procoagulant and anticoagulant factors during pediatric cardiac operations may contribute to the attendant bleeding diathesis or thrombus formation commonly encountered in these patients. The endothelium appears to play an important role in the regulation of hemostasis. The TM-protein C system was significantly more altered in children weighing less than 10 kg. Further studies are necessary to elucidate the exact role of these endotheliumderived substances in the setting of pediatric cardiac procedures and whether they can be positively influenced.
References Bick RL. Hemostasis defects associated with cardiac surgery prosthetic devices and extracorporeal circuits. Semin Thromb Hemost 1985;11:249-80. Gerlach H, Esposito C, Stern D. Modulation of endothelial hemostatic properties: an active role in the host response. Annu Rev Med 1990;41:1524. Wu KK. Endothelial cells in hemostasis, thrombosis, and inflammation. Hosp Pract 1992;April:145-66. Takano S , Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood 1990;76:2024-9.
1589
Ann Thorac Surg 1994;571584-9
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
5. Dittman WA. Thrombomodulin. Biological and cardiovascular applications. Trends Cardiovasc Med 1991;1:3314. 6. Esmon NL. Thrombomodulin. Prog Hemost Thromb 1989;9: 29-55. 7. Ogawa S, Gerlach H, Esposito C, Pasagian-Macaulay A, Brett J, Stern D. Hypoxia modulates the barrier and coagulant function of the cultured bovine endothelium: increased monolayer permeability and induction of procoagulant properties. J Clin Invest 1990;85:1090-8. 8. Amiral J, Adam M, Mimilia F, Larrivaz I, Chambrette B, Boffa MC. A new assay for soluble forms of thrombomodulin in plasma. Thromb Haemost 1991;65:947. 9. Manno CS, Hedberg KW, Kim HC, et al. Comparison of the hemostatic effects of fresh whole blood, stored whole blood, and components after open heart surgery in children. Blood 1991;5:93W. 10. Wyss M, Babel JF, Rouge JC, Bouvier CA. Haemostatic changes during open heart surgery with extracorporeal circulation and deep hypothermia in children. Anaesthesist 1982;31:82-6. 11. Thompson EA, Salem HH. The role of thrombomodulin in the regulation of hemostatic interactions. Prog Hematoll987; 15:51-70. 12. Mann KG, Krishnaswamy S, Lawson JH. Surface-dependent hemostasis. Semin Hematol 1992;29:213-26. 13. Boffa MC, Karochkine, Berad M. Plasma thrombomodulin as a marker of endothelium damage. Nouv Rev Fr Hematol 1991;33:529-30. 14. Kodama S, Uchijima E, Nagai M, Mikawatani K, Hayashi T, Suzuki K. One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies. Clin Chim Acta 1990;192:191-9.
15. Aurrousseau MH, Amiral J, Boffa MC. Level of plasma thrombomodulin in neonates in children. Thromb Haemost 1991;65:1232. 16. Tomura S, Nakamura Y, Deguchi F, et al. Plasma vonwillebrand factor and thrombomodulin as markers of vascular disorders in patients undergoing regular hemodialysis therapy. Thromb Res 1990;58:413-9. 17. Gibbs NM, Crawford PM, Michalopoulos N. Postoperative changes in coagulant and anticoagulant factors following abdominal aortic surgery. J Cardiothorac Vasc Anesth 1992; 6:680-5. 18. Asakura H, Jokaji H, Saito M, et al. Plasma levels of soluble thrombomodulin increase in cases of disseminated intravascular coagulation with organ failure. Am J Hematol 1991;38: 281-7. 19. Tanaka K, Wada K, Morimoto T, et al. The role of the protein C-thrombomodulin system in physiologic anticoagulation during cardiopulmonary bypass. ASAIO Trans 1989;35: 37s5. 20. Knob1 PN, Zilla P, Fasol R, Miiller MM, Vukovich TC. The protein C system in patients undergoing cardiopulmonary bypass. J Thorac Cardiovasc Surg 1987;94600-5. 21. Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985;76:217843. 22. Feindt P, Volkmer I, Seyffert UT, Haack H. The role of protein C as an inhibitor of blood clotting during extracorporeal circulation. Thorac Cardiovasc Surg 1991;39:338-43. 23. Griffin JM, Mosher DF, Zimmermann TS, Kleiss AJ. Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood 1982;60:2614. 24. Manucci PM, Vigano S. Deficiencies of protein C, an inhibitor of blood coagulation. Lancet 1982;2:463-7.
In 30 consecutive children with congenital heart disease scheduled for pediatric cardiac operations, thrombomodd i n , protein C, free protein S, and thrombin-antithrombin complex were measured by enzyme-linked immunosorbent assay after the induction of anesthesia (baseline value), and then before, during, and after cardiopulmonary bypass until the first postoperative day. The patients were divided prospectively into two groups: children weighing less than 10 kg (group l; n = 15)and those weighing more than 10 kg (group 2; n = 15). At baseline, the plasma concentration of thrombomodulin was significantly higher in the children in group 1 than in those in group 2 (83.1 & 11.0 ng/mL versus 29.2 & 12.1 ng/mL). During cardiopulmonary bypass, the thrombomodulin level was reduced in both groups without showing any significant group differences. Five hours after cardiopulmonary bypass and on the first postoperative day, the thrombomodulin level exceeded normal values only in the children weighing less than 10 kg. In both groups, the protein C levels were already below normal at the beginning of the study. The baseline protein S concentration was higher in the smaller children (80% & 18%) than in the larger children (66% & 11%). It was reduced
C
ardiopulmonary bypass (CPB) is known to profoundly influence plasma- and platelet-associated coagulation [l].This may result either in prolonged or enhanced bleeding, or both, or in early thrombosis stemming from contact activation by the nonendothelial synthetic surfaces of the extracorporeal circulation equipment. In recent years, it has become obvious that the vascular endothelium is metabolically active, and this includes the regulation of pressure and hemostasis [2, 31. The vascular endothelial cells produce various important antithrombotic substances such as plasmin activators, heparinlike enzymes, and others [3]. Thrombomodulin (TM) is one of these endothelial cell products that contributes to the regulation of coagulation [4-6]. A growing body of data indicates that TM plays an important role in maintaining the anticoagulant surface of the endothelium [6].Expression of TM from the endothelial surface is activated by various stimuli, including low blood flow and hypoxia [7]. Accepted for publication Oct 20, 1993. Address reprint requests to Dr Boldt, Department of Anesthesiology and Intensive Care Medicine, Justus-Liebig-University Giessen, Klinikstrasse 29, 35392 Giessen, Germany.
0 1994 by The Society of Thoracic Surgeons
by cardiopulmonary bypass in both groups; however, postoperatively it did not return to normal in group 1 (45.1% 2 10%). Plasma levels of the thrombinantithrombin complex were similar in both groups, with a marked increase at the end of cardiopulmonary bypass, and returned to near-normal levels by 5 hours after bypass. Other coagulation variables (the partial thromboplastin time and the antithrombin 111 and fibrinogen levels) were not significantly different between the two groups. Postoperative blood loss was significantly higher in the smaller children on the first postoperative day (35.3 & 20.1 mL/kg) than in the children weighing more than 10 kg (11.8 2 5.0 mL/kg). Five children in group 1 and 2 children in group 2 received fresh frozen plasma in the postbypass period (p < 0.05). It is concluded that the endothelium is involved in the regulation of hemostasis by producing the natural anticoagulant thrombomodulin. This endothelium-related system was significantly more altered in the smaller children with congenital heart disease undergoing cardiac operations, than it was in the bigger children, which may have contributed to the greater bleeding tendency in these children. (Ann Thorac Surg 1994;57:1584-9)
Thrombin binds to TM, and then this thrombin-TM complex activates protein C, which is known to be a potent anticoagulant. Together with protein S as a cofactor, activated protein C degrades coagulation factors Va and VIIIa, thereby limiting clotting activity [3, 51. Thus, abnormalities in TM production may result in abnormal regulation of coagulation [6]. Only very little is known of this complex interplay between endothelium and coagulation in patients suffering from congenital heart disease. Thus, the present study was carried out to investigate changes in the plasma levels of TM in pediatric patients undergoing cardiac operations using CPB.
Material and Methods Pa tients Thirty consecutive children scheduled for palliative or corrective operations for the treatment of congenital heart disease were included in this prospectively designed study. Informed consent was obtained from the parents according to the protocol of the ethics study committee of the hospital. Exclusion criteria were a reoperation, use of anticoagulant drugs within 10 days before the operation (heparin, cyclooxygenase inhibitors), and renal insuffi0003-4975/94/$7.00
Ann Thorac Surg 1994;57:1584-9
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
ciency (creatinine level exceeding 1.5 mg/dL). The children were divided into two groups according to weight. Group 1 consisted of 15 children weighing less than 10 kg; group 2 was made up of 15 children weighing between 10 and 25 kg. The anesthesia regimen was similar for all patients, and consisted of weight-related doses of fentanyl (total dose, 0.05 ? 0.02 mg/kg), midazolam (total dose, 0.65 2 0.03 mg/kg), and pancuronium bromide (total dose, 0.35 k 0.05 mg/kg). All patients were ventilated mechanically at least until the first postoperative day. Ventilation patterns were selected according to pulse oximetry, endexpiratory carbon dioxide, and blood gas analysis values. No volatile anesthetics were used during the investigation period. Anesthesia was always administered by the same anesthesiologist, who was not involved in the study.
Measured Variables and Data Points
Cardiopulmonary Bypass Five minutes before the start of CPB, 300 U/kg of bovine heparin preparation was administered to achieve anticoagulation. The activated clotting time was kept at greater than 400 seconds during the entire bypass period. A COBE VPCMLplus membrane oxygenator (Cobe Laboratories, Lakewood, CO) was used, and a flow of 2.4 L/min * m2 was maintained during CPB. Priming of the extracorporeal circuit consisted of Ringer's solution, 5% human albumin, and electrolytes. One unit of packed red blood cells was added to the prime in all children weighing less than 10 kg. In the group of children weighing more than 10 kg, packed red blood cells were added with regard to the preoperative hematocrit value (packed red blood cells were added when the preoperative hematocrit was less than 0.35). The amount of the priming volume was identical in all children (800 mL). Additional packed red blood cells were given when the hematocrit was less than 0.20. When necessary, Ringer's solution was added to maintain the filling of the circuit. After the end of CPB, the blood remaining in the extracorporeal oxygenation equipment was salvaged by a centrifugation device (Cell Saver 111; Haemonetics, Braintree, MA). All autologous blood was retransfused in the postbypass period. Heparin was neutralized by the administration of protamine chloride in a ratio of 1:l with regard to the initially given dose of heparin. Further protamine was given when the activated clotting time exceeded 200 seconds. All children were operated on by the same surgeon. The decision to administer packed red blood cells and blood derivatives (fresh frozen plasma, platelet concentrates) was made by physicians who were not involved in the study. Packed red blood cells were given when the hematocrit was less than 0.30. After careful exclusion of surgical bleeding, administration of fresh frozen plasma was indicated postoperatively when bleeding exceeded 5 mL * kg-' * h-' and the platelet count exceeded 50,00O/mL (activated clotting time, <200 seconds). Platelet concentrates were administered when bleeding exceeded 5 mL * kg-' h-' and the platelet count was less than 50,00O/mL (activated clotting time, (200 seconds).
1585
Hemoglobin, hematocrit, and standard coagulation variables (activated clotting time, antithrombin 111, and fibrinogen plasma concentrations, as well as the partial thromboplastin time were determined in arterial blood samples. The TM plasma levels were measured by a one-step sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody (Diagnostica Stago, Asnieres Cedex, France) raised against human TM [8]. The ELISA used two monoclonal antibodies that react at different sites of the TM molecule and do not interfere with each other's binding. Protein C and S plasma concentrations were also assessed by ELISA (Boehringer-Mannheim, Mannheim, Germany). Protein S was quantified as free protein S after bound protein S was removed from the plasma by precipitation with polyethylene (normal for free protein S, >30%). Normal values of TM in healthy volunteers determined with this method were reported to be less than 40 ng/mL [lo]; the protein C concentration normally ranged from 70% to 110%. The thrombinantithrombin complex was also measured by ELISA (Behringwerke, Marburg, Germany [normal values, <30-40 pg/L]). All data from the ELISAs represent the means of duplicate measurements. Measurements were performed after induction of anesthesia (the baseline values), before the start of CPB (before anticoagulation with heparin), 20 minutes after the onset of CPB, immediately after the end of CPB (before infusion of protamine), at the end of operation, 5 hours after the end of CPB, and on the morning of the first postoperative day. The quantities of fluids administered (including fresh frozen plasma and packed red blood cells) were recorded and the urine output, blood loss from postbypass suction, and chest tube drainage were also documented.
Stat is t ics Mean values and standard deviations were calculated for all variables. Statistical interpretation was performed by multivariate (repeated-measures) analysis of variance (including multiple-range tests [Scheffcs test]). 2 analysis was performed for differences in homologous blood use. Values of p less than 0.05 were considered significant.
Results The patients' characteristics and the data from CPB are shown in Table 1.The children in group 1 ( < l o kg) ranged in age from 3 days to 13 months and their body weight ranged from 3.7 to 7.9 kg. The children in group 2 (>lo kg) ranged in age from 35 to 68 months and their weight ranged from 13.3 to 22.5 kg. There were no differences among the groups with regard to the duration of the bypass period, the lowest temperatures during bypass, and the number of cyanotic and acyanotic congenital heart diseases. The breakdown of the surgical procedures was similar for both groups (Table 2). The degree of hemodilution (hemoglobin) and the values for standard coagulation variables (antithrombin 111, fibrinogen, partial thromboplastin time, platelet count)
1586
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
Table 1 . Patients’ Characteristics and Data From Cardiopulmonaru Bupass Patient Characteristics Age (mo) Weight (kg) Cyanotic Acyanotic CPB (min) Ischemia”(min) Total heparin (units) Lowest temperature (“C) Rectal Nasopharyngeal Tube drainage (mLkg) 5 hr after CPB 1st postoperative day Use of homologous blood and products (except PRC added to the prime) (mL) PRC FFP a
Period of aortic cross-clamping.
CPB = cardiopulmonary bypass; = packed red blood cells.
Group 1 (
Group 2 (1 ’0
kg)
7.0 f 3.3 5.5 f 1.0 6 9 157 f 55 95 f 32 2,677 f 641
54.2 ? 10.lb 16.1 ? 2.0b 7 8 154 f 42 87 ? 45 10,532 ? 1,59Bb
30.2 f 4.1 28.3 2 4.4
31.7 ? 2.6 29.0 f 3.7
11.6 2 6.9 35.3 f 20.1
3.4 2 2.1b 11.8 2 5.0b
610 285
230b 150
p < 0.05. FFP = fresh frozen plasma;
PRC
did not differ significantly between the two groups (Table 3). Five hours after CPB and on the first postoperative day, the postoperative blood loss was significantly higher in group 1 than in group 2 (see Table 1). The total use of homologous blood and blood products in the postoperative period was greater in the smaller children (Table 1):6 and 5 children in group 1required packed red blood cells and fresh frozen plasma, respectively, compared to 2 and 2 children, respectively, in group 2 ( p < 0.05). The TM plasma concentration at baseline was significantly higher in group 1 than in group 2 children (Fig 1). The TM concentration was not influenced by the anesthesia or the surgical procedure itself (no change from the level after induction until the level before the start of CPB). During CPB, the TM concentration was reduced in both groups without showing any significant differences between the groups. Five hours after CPB and on the first postoperative day, the TM level exceeded normal values in the group 1 children, but it almost returned to normal in the group 2 children. The protein C level was already below normal in both groups at the beginning of the study (approximately59%) (Fig 2). During CPB and immediately after, the decrease was more pronounced in group 1 children (from 59% to 19%) than in group 2 children (from 59% to 39%). Postoperatively, it remained below normal in both groups without any significant differences between the groups. The protein S level was higher at baseline in the smaller children (80%), although it was normal in the children weighing more than 10 kg (66%) (see Fig 2). In children
Ann Thorac Surg 1994;571584-9
weighing less than 10 kg (group l), the protein S plasma level did not return to the baseline level postoperatively (45.1% -C 10%). The thrombin-TM complex values showed a similar course in both groups, with a marked increase at the end of CPB and at the end of the operation (Fig 3). The values had almost normalized by 5 hours after the end of CPB in both groups.
Comment Coagulation abnormalities that occur in children undergoing pediatric cardiac operations represent a complex interrelationship between preexisting coagulation defects and coagulation defects secondary to CPB [9]. Wyss and associates [lo] reported that 11 of the 20 children in their study who underwent cardiac operations exhibited various coagulation problems preoperatively. After CPB, additional alterations in the coagulation status were observed. In addition to platelet-related and factor-associated coagulation abnormalities, endothelium-derived changes constitute a new dimension as a source of disturbances in coagulation. Thrombomodulin is a surface protein expressed on the endothelium that is involved in the regulation of coagulation. It neutralizes thrombin clotting activity and accelerates the thrombin-catalyzed activation of protein C [3, 5, 111. Thus, together with protein C, it represents an important physiologic anticoagulant system 13, 121. One major finding in the present study was that, after the induction of anesthesia (before CPB), the TM level was significantly higher in the children weighing less than 10 kg than in the children weighing more than 10 kg. During and immediately after CPB, the TM concentration decreased significantly in both groups-most likely due to hemodilution during CPB. Postoperatively, the TM concentration increased beyond normal values only in the group 1 children. Normal TM plasma levels (in healthy adults) are not clearly defined. It appears to be highly dependent on the ELISA method used for measuring the TM concentration. With the method used in the present study, the normal values of TM were found to be 30 to 40 pg/L [13]. Using different assays, other authors have
Table 2. Type of Operation in the Two Groups Type of Operation Fallot IV repair TGA ASD + VSD repair PST + ASD repair PST + VSD repair Aortopulmonary shunt Mitral valve replacement AV canal repair Homograft
Group 1 (
Group 2 (’10 kg)
2 2 1 1 1 2
1 1 2
... 3 2
...
2
2 2
2 2
IV = intravenAV = atrioventricular; ASD = atrial septal defect; PST = stenosis of the pulmonary artery; TGA = transposition tricular; VSD = ventricular septal defect. of the great arteries;
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
Ann Thorac Surg 1994;57:15849
1587
Table 3. Hemoglobin, Antithrombin III, and Fibrinogen Plasma Levels and the Partial Thromboplastin Time in the Two Groups Hematologic Variable
After Induction
Before CPB
Hemoglobin (g/dL) <10 kg >10 kg Antithrombin 111 (%)”
14.0 f 1.1 12.7 t 2.1
13.4 & 0.9 12.2 f 2.2
77 +. 12 80 2 11
77 & 10 79 t 10
199 2 22 220 2 24
195 t 20 205 f 21
>10 kg Fibrinogen (g/L)b 4 0 kg >10 kg PTT (s)c 4 0 kg >10 kg Normal values: a 70%-110%; CBP
=
50 52
50 f 11 54 2 10 1 . 5 4 0 g/L;
cardiopulmonary bypass;
p.0. =
f f
After CPB
End of Operation
5 Hours after CPB
1st p.0. Day
10.4 f 1.1 10.1 t 1.9
10.3 f 0.7 11.9 f 1.4
12.3 t 1.8 12.6 +- 2.2
13.7 f 1.9 12.5 t 1.8
50 f 11 56 t 11
53 5 8 49 f 10
49 2 8 56 & 12
8 9 f 11 90 f 13
98 2 12 107 & 14
109 & 22 125 f 29
>300 >300
>300 >300
During CPB 8.3 9.2
11 9
f &
0.7 0.9
10 9
58 t 11 60 2 9
142 t 22 139 & 28
253 t 32 287 f 33
12 11
58 & 11 48 2 14
60 59
64 f 11 51 ? 12
56 46
f f
f f
25-35 seconds.
PTT = partial thromboplastic time
postoperative;
separated children according to weight because the child’s weight appears to be more closely related to maturity than the child’s age. Increased levels of TM may either result from (generalized) endothelial alterations or kidney dysfunction [3, 161. As renal insufficiency was ruled out by our exclusion
6o
50 40
-
-
30
I (ng/ml 110 100
I00
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
--
*
:
----
protein S
(%)
1
10 0
I
I
after induction
I
before CPB
I
CPB
I
after CPB
I
I
I
end of 5 hrs 1. p.0 operation after CPB day
Fig 1. Thrombomodulin plasma levels (in nanograms per milliliter) in the two groups (normal values in the adult, < 3 U O nglmL). (CPB = cardiopulmonary bypass; p.0. = postoperative; SD = standard deviation.)
after before induction CPB
CPB
after CPB
end of
5 hrs
operation after CPB
1 . p.0 day
Fig 2. Protein C and (free) protein S plasma concentrations (normal values in the adult: protein C, 70% to 110%;free protein S, >30%). (CPB = cardiopulmonary bypass; p.0. = postoperative; SD = standard deviation.)
1588
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
240 220 200 I80
160 I40 I20 I00 80 60 40
20
after induction
I
I
I
I
before CPB
CPB
after CPB
-B-
I
I
I
end of 5 hrs I . p.0 operation after CPB day
-0-
>IOkg
I
Fig 3 . Thrombin-antithrombin plasma levels in the two groups (normal values in the adult, <30 pgFL). (CPB = cardiopulmonary bypass; p.0. = postoperative; SD = standard deviation.)
to endothelial cell damage [13]. Hypoxemia may cause alterations in plasma- and platelet-associated coagulation [17]. Hypoxia, however, was reported to lead to a decrease in TM activity on the cell surface [7]. Moreover, there were no differences among the two groups in the present study in terms of the number of cyanotic and acyanotic congenital heart diseases. In the setting of disseminated intravascular coagulation and pulmonary insufficiency, the soluble TM level was reported to be significantly increased [3, 181. In the disseminated intravascular coagulation syndrome, activated proteolytic enzymes in the coagulation-fibrinolysis system may induce release of TM. In the setting of the adult respiratory distress syndrome, proteases released from leukocytes may be responsible for the release of TM into the circulation [4]. The thrombin-antithrombin complex plasma levels (and standard coagulation variables) at the beginning of the study showed no evidence of activation of the coagulation system leading to thrombin generation [ 19, 201. As known for other endothelium-derived substances (eg, endothelin, endothelium-derived releasing factor), the maintenance of blood fluidity and microcirculatory blood flow may also play a central role in maintaining endothelial integrity [6], by which TM expression and release may be influenced. It is difficult to decide whether the smaller (younger) children in the present study were more ill, and thus affected by more altered hemodynamics, than were the children weighing more than 10 kg. It also cannot be definitely concluded from the present results just how far changes in the circulating TM levels represent changes in endothelial expression, and vice versa. One problem with endothelium-associated substances is that they are expressed on the endothelial surface. Although TM is mainly found on endothelial cell surfaces, it is also present in circulating blood [21]. Thrombomodulin also appears to be active in its soluble form: when TM
Ann Thorac Surg 1994;5715%9
was purified from plasma, it was found to catalyze protein C activation by thrombin. The apparent Michaelis constant for protein C was identical for both the soluble and cellular forms of TM. The protein C concentration was most markedly reduced by CPB in the small children. This may be due to an increased activation and increased elimination by the reticuloendothelial system [22]. Lowered plasma levels of protein C have been observed during in vivo activation of the blood coagulation system [23]. In this situation, this may be due to the clearance of protein C activated by thrombin [24]. The protein C level remained lower in the children weighing less than 10 kg than in those weighing more than 10 kg until the end of the investigation period. In adults undergoing cardiac procedures, Knob1 and associates [20] also found a decreased protein C plasma concentration during and after CPB. They assumed that this might be one reason for the well-known bleeding tendency seen in patients undergoing cardiac procedures. One explanation for the reduced protein C levels in the small children ( < l o kg) in the present study might be a failure to adequately synthesize the quantities necessary to replenish the protein C losses that occur during CPB. Bleeding was greater in our group 1 children and the altered TM-protein C system may additionally contribute to this phenomenon. The baseline protein S plasma levels were also significantly lower in the smaller children, and were still reduced in the postoperative period. This agrees with observations made in adults, who showed a significant decrease in the TM, protein C, and protein S plasma concentrations during CPB [20]. This decrease is considered to reflect the activation and consumption of the TM-protein C system in response to thrombin generation. The decrease in the present study, however, was much more pronounced than that seen in adult patients undergoing cardiac procedures. It is concluded from our findings that the imbalance in procoagulant and anticoagulant factors during pediatric cardiac operations may contribute to the attendant bleeding diathesis or thrombus formation commonly encountered in these patients. The endothelium appears to play an important role in the regulation of hemostasis. The TM-protein C system was significantly more altered in children weighing less than 10 kg. Further studies are necessary to elucidate the exact role of these endotheliumderived substances in the setting of pediatric cardiac procedures and whether they can be positively influenced.
References Bick RL. Hemostasis defects associated with cardiac surgery prosthetic devices and extracorporeal circuits. Semin Thromb Hemost 1985;11:249-80. Gerlach H, Esposito C, Stern D. Modulation of endothelial hemostatic properties: an active role in the host response. Annu Rev Med 1990;41:1524. Wu KK. Endothelial cells in hemostasis, thrombosis, and inflammation. Hosp Pract 1992;April:145-66. Takano S , Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood 1990;76:2024-9.
1589
Ann Thorac Surg 1994;571584-9
BOLDT ET AL THROMBOMODULIN AND PEDIATRIC CARDIAC SURGERY
5. Dittman WA. Thrombomodulin. Biological and cardiovascular applications. Trends Cardiovasc Med 1991;1:3314. 6. Esmon NL. Thrombomodulin. Prog Hemost Thromb 1989;9: 29-55. 7. Ogawa S, Gerlach H, Esposito C, Pasagian-Macaulay A, Brett J, Stern D. Hypoxia modulates the barrier and coagulant function of the cultured bovine endothelium: increased monolayer permeability and induction of procoagulant properties. J Clin Invest 1990;85:1090-8. 8. Amiral J, Adam M, Mimilia F, Larrivaz I, Chambrette B, Boffa MC. A new assay for soluble forms of thrombomodulin in plasma. Thromb Haemost 1991;65:947. 9. Manno CS, Hedberg KW, Kim HC, et al. Comparison of the hemostatic effects of fresh whole blood, stored whole blood, and components after open heart surgery in children. Blood 1991;5:93W. 10. Wyss M, Babel JF, Rouge JC, Bouvier CA. Haemostatic changes during open heart surgery with extracorporeal circulation and deep hypothermia in children. Anaesthesist 1982;31:82-6. 11. Thompson EA, Salem HH. The role of thrombomodulin in the regulation of hemostatic interactions. Prog Hematoll987; 15:51-70. 12. Mann KG, Krishnaswamy S, Lawson JH. Surface-dependent hemostasis. Semin Hematol 1992;29:213-26. 13. Boffa MC, Karochkine, Berad M. Plasma thrombomodulin as a marker of endothelium damage. Nouv Rev Fr Hematol 1991;33:529-30. 14. Kodama S, Uchijima E, Nagai M, Mikawatani K, Hayashi T, Suzuki K. One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies. Clin Chim Acta 1990;192:191-9.
15. Aurrousseau MH, Amiral J, Boffa MC. Level of plasma thrombomodulin in neonates in children. Thromb Haemost 1991;65:1232. 16. Tomura S, Nakamura Y, Deguchi F, et al. Plasma vonwillebrand factor and thrombomodulin as markers of vascular disorders in patients undergoing regular hemodialysis therapy. Thromb Res 1990;58:413-9. 17. Gibbs NM, Crawford PM, Michalopoulos N. Postoperative changes in coagulant and anticoagulant factors following abdominal aortic surgery. J Cardiothorac Vasc Anesth 1992; 6:680-5. 18. Asakura H, Jokaji H, Saito M, et al. Plasma levels of soluble thrombomodulin increase in cases of disseminated intravascular coagulation with organ failure. Am J Hematol 1991;38: 281-7. 19. Tanaka K, Wada K, Morimoto T, et al. The role of the protein C-thrombomodulin system in physiologic anticoagulation during cardiopulmonary bypass. ASAIO Trans 1989;35: 37s5. 20. Knob1 PN, Zilla P, Fasol R, Miiller MM, Vukovich TC. The protein C system in patients undergoing cardiopulmonary bypass. J Thorac Cardiovasc Surg 1987;94600-5. 21. Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985;76:217843. 22. Feindt P, Volkmer I, Seyffert UT, Haack H. The role of protein C as an inhibitor of blood clotting during extracorporeal circulation. Thorac Cardiovasc Surg 1991;39:338-43. 23. Griffin JM, Mosher DF, Zimmermann TS, Kleiss AJ. Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood 1982;60:2614. 24. Manucci PM, Vigano S. Deficiencies of protein C, an inhibitor of blood coagulation. Lancet 1982;2:463-7.