American Journal of Hematology 38:86-89 (1991)
Dynamic Fluctuations in Blood of Thrombin/ Antithrombin 111 Complex (TAT) Katsumi Deguchi, Mituo Noguchi, Eiichi Yuwasaki, Takurou Endou, Akira Deguchi, Hideo Wada, Seikou Murashima, Masakatu Nishikawa, Shigeru Shirakawa, Kuniyoshi Tanaka, and Minoru Kusagawa Second Department of Internal Medicine (K.D., M.N., E.Y., T.E., A.D., H.W., S.M., M.N., S.S.) and Department of Thoracic Surgery (K.T., M.K.), School of Medicine, Mie University, Edobashi, Tsu-city, Mie, Japan
The dynamic fluctuation of thrombin-antithrombin 111 complex (TAT) was studied in blood obtained during the daytime (at 9 AM, noon, 3 PM), during extracorporeal circulation and during the course of disseminated intravascular coagulation (DIC), to certify whether the level of TAT in blood can reflect the generation of thrombin. In 10 healthy male volunteers, the mean values of TAT (kg/liter) were 1.74 (51.36) at 9 AM, 1.22 (k0.47) at noon, and 1.25 (50.68) at 3 PM. TAT did not show a daytime fluctuation, unlike fibrinolytic factors. The mean values of TAT in 38 hemodialyzed patients were 4.83 (+-2.8)before the initiation, 6.59 (54.39) in the first hour, and 13.42 (k10.96) at the end of a dialysis session. In 20 patients undergoing open heart surgery, the mean value of TAT was increased during cardiopulmonary bypass (CPB) and decreased with time after the end of surgery. The fibrinopeptide A (FPA) value was increased with TAT during CPB but achieved a maximum level immediately after heparin neutralization by protamine. In 20 patients with DIC, the values of TAT varied from 5.8 to 297 pg/liter in the blood at the onset of DIC. In seven of eight patients treated with low-molecular-weightheparin (LMW-H), the values of TAT and FPA were lower 24 hr after LMW-H than before the treatment. These results suggest that the level of TAT in blood reflected the formation of thrombin and could serve as a sensitive parameter of activated coagulation in circulating blood. Key words: daytime fluctuation, hemodialysis, cardiopulmonary bypass, disseminated intravascular coagulation
INTRODUCTION
The assessment of clotting activation is essential to evaluate hypercoagulable (prethrombotic) and thrombotic states. During the reaction in the blood coagulation cascade, factor Xa cleaves prothrombin, which results in the generation of thrombin. This serine protease can rapidly be neutralized by antithrombin 111 (AT 111), with the formation of inactive thrombin-AT I11 complex (TAT). Furthermore, the release of fibrinopeptide A (FPA) from fibrinogen may follow augmented thrombin generation. Most intermediates, such as TAT and FPA, formed during slight clotting activation in circulating blood occur in very small amounts. However, the measurement of such intermediates has become possible with enzyme-li&ed immunosorbent assays (ELISA). only prepared for the measurement Of was an TAT, which KIdCes possible a direct approach to the study of in vivo thrombin generation [I]. This study 0 1991 Wiley-Liss, Inc.
evaluated the dynamic fluctuation of TAT in blood during the daytime in volunteers, during extracorporeal circulation and during the course of DIC, to certify whether the level of TAT in blood can reflect the generation of thrombin attendant on the activation of coagulation factors. MATERIALS AND METHODS
The subjects consisted of 10 healthy male volunteers (23-30 years old), 38 patients on maintenance hemodialysis, 20 patients undergoing open heart surgery, and 20
Received for publication September 4, 1990; accepted May 16, 1991. Address reprint requests to Katsumi Deguchi, Second Department of Internal Medicine, School of Medicine, Mie University, 2-174 Edobashi, Tsu-city, Mie 514, Japan.
Dynamic Fluctuation in Blood of TAT 25
i %
UG/L
200
5.0
20
I
-=, . -
15
a
r
L
0
2
E
I00
2.5
Ln
+
f 9"
12"
15"
10
5 9"
12"
15"
Fig. 1. Individual changes of TAT as absolute amount (left) and as percentages (right) in blood from 10 volunteers obtained at three times during the day (9.00,12.00 and 15.00 h). Circles show the median values (+standard deviation) of TAT.
patients with disseminated intravascular coagulation (DIC). Blood samples were taken by venepuncture from an antecubital vein under fasting conditions during the daytime at 9 AM,noon, and 3 PM in 10 healthy male volunteers. Sodium cintrate (3.8%) as an anticoagulant was used, and the prepared plasma samples were stored at -80°C until measurement. TAT was determined by solid-phase enzyme immunoassay (Enzygynost-TAT kit; Behringwerke AG, Federal Republic of Germany), and FPA by an RIA PEG method. Heparin was measured by a chromogenic substrate assay with S-2222. Results are expressed as mean standard deviation (SD) and the significance of differences between two groups was assessed by Student's nonpaired t test. +_
RESULTS Alteration in the Level of TAT During the Day
Figure 1 shows individual changes of TAT in blood samples from 10 volunteers obtained at three times during the daytime (9 AM, noon, 3 PM).The values of each individual blood sample obtained at 9 AM ranged from 0.6 to 5.5 @/liter. Only one sample showed a level exceeding the upperlimit of 4.5 pg/liter within the 95% confidence interval for TAT values from 60 healthy subjects [2]. The ranges of change as percentages and as absolute amounts for the noon and 3 PM values relative to those at 9 AM were 20.7-1 17% and 0.4-1.9 pg/liter, 22.4-254.5% and 0.4-2.8 pg/liter, respectively. The mean values (+SD) of TAT (kg/liter) were 1.74 (k 1.36) at 9 AM,1.22 (k0.47) at noon, and 1.25 (k0.68) at 3 PM.
I
Before dialysis
I
1 s t hour of d i a l y s i s
endof dialysis
Fig. 2. Mean value of TAT (?SD)and the incidence of level greater than 4.5 kg/liter TAT in patients (n = 38) on maintenance hemodialysis.
Alteration in Level of TAT During Extracorporeal Circulation
Patients on maintenance hemodialysis. In 38 patients receiving hemodialysis for 6 hr, daily, three times/week, the TAT value in the blood collected immediately before the initiation of dialysis was 1.9-15.5 Fgiliter. The mean value of TAT (LSD) (pgiliter) and the incidence of level greater than 4.5 pg/liter (%) were 4.83 (k2.8) and 42.1 (16 of 38 patients; 16/38) before the initiation, 6.59 (k4.39) and 81.6 (31/38) in the first hour, and 13.42 (k 10.96) and 100 (5/5)at the end of the dialysis session (Fig. 2). The change occurring between the initiation and the first hour was significant ( P < 0.05). Patients undergoing open heart surgery (Fig. 3). Alteration in the levels of TAT and FPA during cardiopulmonary bypass (CPB) in 20 patients undergoing open heart surgery were studied. Heparinization was obtained with an initial dose of sodium heparin of 300 Uikg immediately before vena cava cannulation, and was monitored by the ACT value, which was kept equal to or greater than 400 sec throughout CPB . At the end of CPB, residual circulating heparin was neutralized by protamine sulfate, in a ratio of 1:1.5 of the total amount of heparin administered. These parameters were expressed in Htcorrected values. The mean value of TAT was increased during CPB and decreased with time after the end of surgery. The FFA value was increased with TAT during CPB but achieved a maximum level immediately after heparin neutralization by protamine.
-
-
Deguchi et al.
88
HEPAR IN
-
TABLE 1. New Standard for DIC Diagnosis'
PROTAMIN
Score
I Basic diseases Exist Do not exist I1 Clinical appearance Bleeding Exist Do not exist Abnormality in internal organs Exist Do not exist I11 Test results FDP in serum (pg/ml)
40
. J
I
0
20 Fk 10
I00
>40
-.
80
_I
a
g 60
. d
3 40 + + 4 20
€-I
' PRE-OP
5
30
60
CARDIOPUMONARY
90
. -
120
BYPASSKPB~
~~(MINI
5 AFTER
CPB
Fig. 3. Alteration of TAT and FPA during cardiopulmonary bypass in 20 patients undergoing open heart surgery. These parameters were expressed Ht-corrected. Circles show the median values (rstandard deviation). P < *0.05, **0.01 vs. preoperative level.
IV
V
Alteration in the Level of TAT During the Course of DIC
In 20 patients who had been diagnosed as having DIC according to the criteria proposed by the research committee on DIC of the Ministry of Health and Welfare in Japan (Table I), TAT values varied from 5.8 to 297 pgiliter in the blood at the onset of DIC. In seven of eight patients treated with low-molecular-weight heparin (LMW-H), the values of TAT and FPA were lower at 24 hr after LMW-H than before treatment (Fig. 4). DISCUSSION
In the present study, the individual values for TAT in 10 healthy male volunteers showed interindividual variation and some variability with time. There were no significant correlations between the mean value of TAT at 9 AM and at noon or 3 PM. This finding suggests that the appearance into the blood of TAT did not show the daytime fluctuations unlike fibrinolytic factors such as
VI
VII
20-40 10-20
Dynamic Fluctuations in Blood of Thrombin/ Antithrombin 111 Complex (TAT) Katsumi Deguchi, Mituo Noguchi, Eiichi Yuwasaki, Takurou Endou, Akira Deguchi, Hideo Wada, Seikou Murashima, Masakatu Nishikawa, Shigeru Shirakawa, Kuniyoshi Tanaka, and Minoru Kusagawa Second Department of Internal Medicine (K.D., M.N., E.Y., T.E., A.D., H.W., S.M., M.N., S.S.) and Department of Thoracic Surgery (K.T., M.K.), School of Medicine, Mie University, Edobashi, Tsu-city, Mie, Japan
The dynamic fluctuation of thrombin-antithrombin 111 complex (TAT) was studied in blood obtained during the daytime (at 9 AM, noon, 3 PM), during extracorporeal circulation and during the course of disseminated intravascular coagulation (DIC), to certify whether the level of TAT in blood can reflect the generation of thrombin. In 10 healthy male volunteers, the mean values of TAT (kg/liter) were 1.74 (51.36) at 9 AM, 1.22 (k0.47) at noon, and 1.25 (50.68) at 3 PM. TAT did not show a daytime fluctuation, unlike fibrinolytic factors. The mean values of TAT in 38 hemodialyzed patients were 4.83 (+-2.8)before the initiation, 6.59 (54.39) in the first hour, and 13.42 (k10.96) at the end of a dialysis session. In 20 patients undergoing open heart surgery, the mean value of TAT was increased during cardiopulmonary bypass (CPB) and decreased with time after the end of surgery. The fibrinopeptide A (FPA) value was increased with TAT during CPB but achieved a maximum level immediately after heparin neutralization by protamine. In 20 patients with DIC, the values of TAT varied from 5.8 to 297 pg/liter in the blood at the onset of DIC. In seven of eight patients treated with low-molecular-weightheparin (LMW-H), the values of TAT and FPA were lower 24 hr after LMW-H than before the treatment. These results suggest that the level of TAT in blood reflected the formation of thrombin and could serve as a sensitive parameter of activated coagulation in circulating blood. Key words: daytime fluctuation, hemodialysis, cardiopulmonary bypass, disseminated intravascular coagulation
INTRODUCTION
The assessment of clotting activation is essential to evaluate hypercoagulable (prethrombotic) and thrombotic states. During the reaction in the blood coagulation cascade, factor Xa cleaves prothrombin, which results in the generation of thrombin. This serine protease can rapidly be neutralized by antithrombin 111 (AT 111), with the formation of inactive thrombin-AT I11 complex (TAT). Furthermore, the release of fibrinopeptide A (FPA) from fibrinogen may follow augmented thrombin generation. Most intermediates, such as TAT and FPA, formed during slight clotting activation in circulating blood occur in very small amounts. However, the measurement of such intermediates has become possible with enzyme-li&ed immunosorbent assays (ELISA). only prepared for the measurement Of was an TAT, which KIdCes possible a direct approach to the study of in vivo thrombin generation [I]. This study 0 1991 Wiley-Liss, Inc.
evaluated the dynamic fluctuation of TAT in blood during the daytime in volunteers, during extracorporeal circulation and during the course of DIC, to certify whether the level of TAT in blood can reflect the generation of thrombin attendant on the activation of coagulation factors. MATERIALS AND METHODS
The subjects consisted of 10 healthy male volunteers (23-30 years old), 38 patients on maintenance hemodialysis, 20 patients undergoing open heart surgery, and 20
Received for publication September 4, 1990; accepted May 16, 1991. Address reprint requests to Katsumi Deguchi, Second Department of Internal Medicine, School of Medicine, Mie University, 2-174 Edobashi, Tsu-city, Mie 514, Japan.
Dynamic Fluctuation in Blood of TAT 25
i %
UG/L
200
5.0
20
I
-=, . -
15
a
r
L
0
2
E
I00
2.5
Ln
+
f 9"
12"
15"
10
5 9"
12"
15"
Fig. 1. Individual changes of TAT as absolute amount (left) and as percentages (right) in blood from 10 volunteers obtained at three times during the day (9.00,12.00 and 15.00 h). Circles show the median values (+standard deviation) of TAT.
patients with disseminated intravascular coagulation (DIC). Blood samples were taken by venepuncture from an antecubital vein under fasting conditions during the daytime at 9 AM,noon, and 3 PM in 10 healthy male volunteers. Sodium cintrate (3.8%) as an anticoagulant was used, and the prepared plasma samples were stored at -80°C until measurement. TAT was determined by solid-phase enzyme immunoassay (Enzygynost-TAT kit; Behringwerke AG, Federal Republic of Germany), and FPA by an RIA PEG method. Heparin was measured by a chromogenic substrate assay with S-2222. Results are expressed as mean standard deviation (SD) and the significance of differences between two groups was assessed by Student's nonpaired t test. +_
RESULTS Alteration in the Level of TAT During the Day
Figure 1 shows individual changes of TAT in blood samples from 10 volunteers obtained at three times during the daytime (9 AM, noon, 3 PM).The values of each individual blood sample obtained at 9 AM ranged from 0.6 to 5.5 @/liter. Only one sample showed a level exceeding the upperlimit of 4.5 pg/liter within the 95% confidence interval for TAT values from 60 healthy subjects [2]. The ranges of change as percentages and as absolute amounts for the noon and 3 PM values relative to those at 9 AM were 20.7-1 17% and 0.4-1.9 pg/liter, 22.4-254.5% and 0.4-2.8 pg/liter, respectively. The mean values (+SD) of TAT (kg/liter) were 1.74 (k 1.36) at 9 AM,1.22 (k0.47) at noon, and 1.25 (k0.68) at 3 PM.
I
Before dialysis
I
1 s t hour of d i a l y s i s
endof dialysis
Fig. 2. Mean value of TAT (?SD)and the incidence of level greater than 4.5 kg/liter TAT in patients (n = 38) on maintenance hemodialysis.
Alteration in Level of TAT During Extracorporeal Circulation
Patients on maintenance hemodialysis. In 38 patients receiving hemodialysis for 6 hr, daily, three times/week, the TAT value in the blood collected immediately before the initiation of dialysis was 1.9-15.5 Fgiliter. The mean value of TAT (LSD) (pgiliter) and the incidence of level greater than 4.5 pg/liter (%) were 4.83 (k2.8) and 42.1 (16 of 38 patients; 16/38) before the initiation, 6.59 (k4.39) and 81.6 (31/38) in the first hour, and 13.42 (k 10.96) and 100 (5/5)at the end of the dialysis session (Fig. 2). The change occurring between the initiation and the first hour was significant ( P < 0.05). Patients undergoing open heart surgery (Fig. 3). Alteration in the levels of TAT and FPA during cardiopulmonary bypass (CPB) in 20 patients undergoing open heart surgery were studied. Heparinization was obtained with an initial dose of sodium heparin of 300 Uikg immediately before vena cava cannulation, and was monitored by the ACT value, which was kept equal to or greater than 400 sec throughout CPB . At the end of CPB, residual circulating heparin was neutralized by protamine sulfate, in a ratio of 1:1.5 of the total amount of heparin administered. These parameters were expressed in Htcorrected values. The mean value of TAT was increased during CPB and decreased with time after the end of surgery. The FFA value was increased with TAT during CPB but achieved a maximum level immediately after heparin neutralization by protamine.
-
-
Deguchi et al.
88
HEPAR IN
-
TABLE 1. New Standard for DIC Diagnosis'
PROTAMIN
Score
I Basic diseases Exist Do not exist I1 Clinical appearance Bleeding Exist Do not exist Abnormality in internal organs Exist Do not exist I11 Test results FDP in serum (pg/ml)
40
. J
I
0
20 Fk 10
I00
>40
-.
80
_I
a
g 60
. d
3 40 + + 4 20
€-I
' PRE-OP
5
30
60
CARDIOPUMONARY
90
. -
120
BYPASSKPB~
~~(MINI
5 AFTER
CPB
Fig. 3. Alteration of TAT and FPA during cardiopulmonary bypass in 20 patients undergoing open heart surgery. These parameters were expressed Ht-corrected. Circles show the median values (rstandard deviation). P < *0.05, **0.01 vs. preoperative level.
IV
V
Alteration in the Level of TAT During the Course of DIC
In 20 patients who had been diagnosed as having DIC according to the criteria proposed by the research committee on DIC of the Ministry of Health and Welfare in Japan (Table I), TAT values varied from 5.8 to 297 pgiliter in the blood at the onset of DIC. In seven of eight patients treated with low-molecular-weight heparin (LMW-H), the values of TAT and FPA were lower at 24 hr after LMW-H than before treatment (Fig. 4). DISCUSSION
In the present study, the individual values for TAT in 10 healthy male volunteers showed interindividual variation and some variability with time. There were no significant correlations between the mean value of TAT at 9 AM and at noon or 3 PM. This finding suggests that the appearance into the blood of TAT did not show the daytime fluctuations unlike fibrinolytic factors such as
VI
VII
20-40 10-20
