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The effect of operation and subcutaneous heparin on plasma levels of antithrombin-iii
THROMBOSIS RESEARCH 43; 657-662, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
THE EFFECT OF OPERATION AND SUBCUTANEOUS HEPARIN ON PLASMA LEVELS OF ANTITHROMBIN-III
Tikva Harhourne,
A.N. Nicolaides,
Academic
Surgical Unit and Irvine Laboratory for Cardiovascular Investigation and Research, St Mary's Hospital Medical School, London, W2, United Kingdom
(Received 19.11.1985; Accepted in revised form 2.5.1986 by Editor J.R. O'Brien) (Received in final form by Executive Editorial Office 30.6.1986) ABSTRACT Plasma antithrombin III (AT-III) levels were measured in 61 The patients were randomly patients undergoing general surgery. allocated to a test or a control group. The test group received 5000iu of subcutaneous heparin two hours before operation and then twelve hourly for seven days. The control group received subPlasma levels of AT-III and of heparin were cutaneous saline. estimated before operation, at two, four and six hours after heparin injection on the day of operation and thereafter before the morning injection on the first, third and sixth postoperative days. Antithromhin III decreased progressively during and after operation in both groups. The decrease was greater in the test group at six These findings support the hours after the first injection. hypothesis that AT-III is consumed during coagulation and its utilization is increased in the presence of heparin.
INTRODUCTION Antithrombin-III is a natural inhibitor to thrombin and factor X and is consumed during coagulation. Heparin is known to potentiate its tgrombin inhibiting capacity presumably due to conformational changes (1). Low levels of AT-III have been implicated in a variety of thrombotic disorders, including those associated with oral contraceptive agents (2,3), venous thromboembolism (4,5) and postoperative myocardial infarction (4,6). Several studies have been done with regard to AT-III levels after surgical procedures. However, the results are conflicting, with some investigators reporting a fall in AT-III (7-g), while other find no significant -.__. Key Words: Antithrombin
III, Subcutaneous 657
Heparin,
Surgery
AT-III
658
HEPARIN & OPERATION
Vol. 43, No. 6
change
(10,ll). Furthermore, conflicting results have been found by several studies of AT-III levels in patients treated with subcutaneous heparin (12,13).
Our study was designed to assess the effect of operation and subcutaneous heparin on the plasma levels of AT-III.
MATERIAL AND METHODS Sixty-one patients over the age of 40 having elective general surgical operations were admitted to the study. They were randomly allocated to a control (33 patients) and a test (28 patients) group. Patients in the test group received 0.2ml of sodium heparin (5,OOOiu), two hours before operation and then twelve-hourly for seven days using the method of Griffith and Boggs (1964) (14). The patients in the control group received 0.2ml of isotonic saline which was administered in the same manner. Collection of blood samples. 4.5ml of titrated blood was collected from each patient just before the pre-operative subcutaneous injection of heparin, then two, four and six hours subsequently on the day of operation. Blood was also collected just before and two hours after the morning injection on the first, third, and sixth postoperative days. The samples were centrifuged immediately at 2,500g for 30 minutes at 4"C, and 2ml of the supernatant platelet free plasma was removed and stored at -2O'C. All samples from each patient were assayed simultaneously. The AT-III assay was carried out on all samples except before heparin injection on the first, 3rd and 6th post operative day. It was carried out on M-Partigen immunodiffusion plates (Hoechst-Behring, Frankfurt, W. Germany) (15), which contain monospecific antiserum against the plasma protein-antithrombin III, in an agar-gel layer. The AT-III in the plasma forms a precipitin ring when in contact with the antibody in the agar. Antithrombin-III concentration is directly proportional to the square of the ring diameter. Protein standard plasma was used for calibration and the normal range obtained in our laboratory was 23-37mg%. The plasma heparin levels were estimated on all samples using a sensitive assay (16) based on the potentiating effect of heparin on the antifactor X . Reproducibility studies in our laboratory have shown that this assay coulda detect changes in heparin levels of O.O2u/ml of plasma.
RESULTS The mean levels of AT-III of the patients in both groups during the six hours after the first injection is shown in Figure 1. Figure 2 shows the mean levels of AT-III at 6 hours after injection on the day of operation and at 2 hours after injection on the first, third and sixth postoperative days. In the levels
control group, the maximum fall in the mean of plasma AT-III compared to pre-operation levels was reached on the third postoperative
Vol. 43, No. 6
AT-III HEPARIN & OPERATION
---
Control group II ~33
-
Test group
659
n-28
Compared to premop levels mpc 35,
---
Control group “=33
-
Test group
0.05
n=28
Compared to pre-op levels
@ D< 0.01
BP” 0.05 opt
31 Mean plasma At-Ill level mg/lOOml 29
0.01
I p< 0.05
35r
33
33
31
T
Mean plasma At-Ill level 29 mg/lOOml I’
25
21
23
25
21
23
\,:/
I
19 I
21I
I 0
J
0
2
4
6
I 1
3 Days
Hours
FIG.
FIG.
1
Mean + SD of AT-III in both groups during the six hours after first injection on the day of Paired t test was used. operation.
6
2
Mean + SD of AT-III at 6 hours after-the first injection of the day of operation, and at 2 hours after injection on the third and sixth postoperative day.
In only two of the 33 controls the level fell below the normal day (Fig 2). the maximum fall was at six hours after the first range. In the test group, injection, ie 2-3 hours after the end of operation (Fig 1). The level fell to This was the only time that a below the normal range in 7 of the 28 patients. significant difference was found when the means of the two groups were compared using a t-test.
jection AT-III
A wide variation in the plasma heparin levels There was no correlation between (Fig 3). levels at any time.
was found after each inthe heparin levels and the
660
AT-III
HEPARIN & OPERATION
Vol. 43, No. 6
.3a ,28 .26 .24 Plasma ‘22 hepark .20 units/ml .,6 % A4 .12 A0 ,08 .06 .04 .02 ,O 0246
02
Operation
Days
FIG. Median and 90 per and after 5,000 morning
02
1S.t
0
3rd
Zhours
6th
postoperative
3
cent tolerance level of heparin levels in the plasma units of subcutaneous heparin on the day of operation third and sixth postoperative day. of the first,
before and
DISCUSSION The results indicate that the plasma level of AT-III was decreased during and after operation in both the control and test groups, with a maximum fall in AT-III level was at six hours after operation. However, the decrease significantly greater in the test group than in the control group. This is the only time heparin has a marked effect on the consumption of AT-III. As this is the time of maximum risk of thrombosis, this finding might well explain why low dose heparin is effective as prophylaxis. The AT-III level during surgery has been studied by several igators; some reported a fall in the plasma AT-III level during (7-9,17); others found no significant changes (10,ll).
investoperation
Our results are in accord with previous reports in which sub-subcutaneous heparin given to patients with deep venous thrombosis and/or pulmonary embolism (18) and to patients with acute myocardial infarction (13)) induced a significant fall in the plasma level of AT-III. They support the hypothesis that AT-III is consumed during coagulation and its utilization is increased in the presence of heparin. Conflicting results have been shown by Kakkar (12) who reported increased AT-III values after subcutaneous heparin that was given to patients withextensive deep venous thrombosis using the same method for measuring plasma ATIII levels, as in our study. These findings (12) are contradicted by the findings of other workers (13,18) as well as our own data.
Vol. 43, No. 6
AT-III
661
HEPARIN & OPERATION
Other studies have investigated the effect of a continuous infusion of iv heparin (19,20) which has been shown to induce a decrease in the plasma levels of AT-III. The levels returned to normal 2-3 days after the discontinuation However, a single iv bolus of heparin did not seem to affect the of heparin. plasma AT-III level (20). It seems that heparin must be present in the blood for several hours in order to reduce AT-III concentrations. Studies on patients with congenital deficiencies of AT-III have shown that low levels of this inhibitor predispose to the development of thromboembolic episodes (21,22). It has been suggested that the low levels of AT-III during treatment with subcutaneous heparin may enhance the risk of developing thrombosis in the interval between the stopping of heparin and the return of AT-III levels to the normal range (20). This emphasizes the importance of adequate anticoagulation with oral agents prior to discontinuation of heparin therapy.
REFERENCES 1.
ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem, 248, 6490-6505, 1973
2.
GREIG, H.B.W. and NOTELOWITZ, M. levels. Lancet, I, 412-3, 1975
3.
VON KAULLA, E. and VON KAULLA, K.N. Oral contraceptives thrombin III activity. Lancet, 1, 36, 1970
4.
VON KAULLA, E. and VON KAULLA, K.N. Clin Pathol, 48, 69-80, 1967
5.
FAGERHOL, M.K., ABILDGAARD, U., BERGSJO, P. and JACOBSEN, J.H. Oral contraceptives and low antithrombin III concentration. Lancet, 1, 1175, 1970
6.
COLLINS, G.J., HEYMANN, R.L. and ZAJTCHUK, R. Hypercoagulability in patients with peripheral vascular disease. Am J Surg, 130, 2-6, 1975
7.
SAGAR, S., STAMATAKIS, J.D., THOMAS, D.P. and KAKKAR VV. Oral contraceptives, antithrombin III activity and postoperative deep vein thrombosis. Lancet, 1, 509-511, 1976
8.
STATHAKIS, N., PAPAYANNIS, A.G. and GARDIKAS, C.D. thrombin III concentration. Lancet, 1, 430, 1973
9.
COLLINS, G.J., BARBER, J.A., ZAJTCHUK, R., VANEK, D., MALOGNE, L.A. The effect of operative stress on the coagulation profile. Am J Surg, 133, 612-616, 1977
10.
HEDNER, U. and NILSSON, I.M. Antithrombin Thrombosis Res, 3, 631-641, 1973
11.
DONATI, M.B., VANDENBERGHE, K., MOLLA, A. DE ROO, M. and VERSTRAETE, Postoperative antithrombin III. Lancet, 1, 726-727, 1973
Natural oestrogens
Antithrombin
and antithrombin
III
and low anti-
III and diseases.
Postoperative
Am J
anti-
III in a clinical material.
M.
662
AT-III HEPARIN & OPERATION
Vol. 43, No. 6
12. KAKKAR, V. V., BENTLEY, P.G., SCLJLLY,M.F., MACGREGOR, I.R., JONES, N.A.G. and WEBB, P.J. Antithrombin III and heparin. Lancet, 1, 103-104, 1980 13.
GREEN P.J. Antithrombin III and subcutaneous heparin. 374-375, 1980
Lancet, 1,
14.
GRIFFITH, G.C. and BOGGS, R.P. Cardiol, 14, 39-46, 1964
15.
MANCINI, G., CARBONARA, A.O. and HEREMANS, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Int J Immunochem, 2, 235-254, 1965
16.
DENSON, K.W. and BONNAR, J. The measurement of heparin. A method based on the.potentiation of antifactor X . Thromb Diath Haemorrh (Stuttg), a 30, 471-479, 1973
17.
SEYFER, A.E., SEABER, A.V., DOMBROSE, F.A. and URBANIAK, J.R. Coagulation changes in elective surgery and trauma. Ann Surg, 193, 210-213, 1981
18.
CONARD, J., LECOMPTE, T., HORELLOU, M.H., CAZENAVE, B. and SAMAMA, M. Antithrombin-III in patients treated with subcutaneous or intravenous heparin. Thrombosis Res, 22, 507-511, 1981
19.
MARCINIAK, E. and GOCKERMAN, J.P. Heparin-induced decrease in circulating antithrombin-III. Lancet, II, 581-584, 1977
20.
RAO, A.K, NIEWIAROWSKI, S., GUZZO, J. and DAY H.J. Antithrombin III levels during heparin therapy. Thrombosis Res, 24, 181-186, 1981
21.
EGBERG, 0. Inherited Antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh, 13, 516-530, 1965
22.
MARCINIAK, E., FARLEY, C. H. and DESIMONE, P.A. Familial thrombosis due to antithrombin-III deficiency. Blood, 43, 219-231, 1974
The clinical usage of heparin.
Am J
THE EFFECT OF OPERATION AND SUBCUTANEOUS HEPARIN ON PLASMA LEVELS OF ANTITHROMBIN-III
Tikva Harhourne,
A.N. Nicolaides,
Academic
Surgical Unit and Irvine Laboratory for Cardiovascular Investigation and Research, St Mary's Hospital Medical School, London, W2, United Kingdom
(Received 19.11.1985; Accepted in revised form 2.5.1986 by Editor J.R. O'Brien) (Received in final form by Executive Editorial Office 30.6.1986) ABSTRACT Plasma antithrombin III (AT-III) levels were measured in 61 The patients were randomly patients undergoing general surgery. allocated to a test or a control group. The test group received 5000iu of subcutaneous heparin two hours before operation and then twelve hourly for seven days. The control group received subPlasma levels of AT-III and of heparin were cutaneous saline. estimated before operation, at two, four and six hours after heparin injection on the day of operation and thereafter before the morning injection on the first, third and sixth postoperative days. Antithromhin III decreased progressively during and after operation in both groups. The decrease was greater in the test group at six These findings support the hours after the first injection. hypothesis that AT-III is consumed during coagulation and its utilization is increased in the presence of heparin.
INTRODUCTION Antithrombin-III is a natural inhibitor to thrombin and factor X and is consumed during coagulation. Heparin is known to potentiate its tgrombin inhibiting capacity presumably due to conformational changes (1). Low levels of AT-III have been implicated in a variety of thrombotic disorders, including those associated with oral contraceptive agents (2,3), venous thromboembolism (4,5) and postoperative myocardial infarction (4,6). Several studies have been done with regard to AT-III levels after surgical procedures. However, the results are conflicting, with some investigators reporting a fall in AT-III (7-g), while other find no significant -.__. Key Words: Antithrombin
III, Subcutaneous 657
Heparin,
Surgery
AT-III
658
HEPARIN & OPERATION
Vol. 43, No. 6
change
(10,ll). Furthermore, conflicting results have been found by several studies of AT-III levels in patients treated with subcutaneous heparin (12,13).
Our study was designed to assess the effect of operation and subcutaneous heparin on the plasma levels of AT-III.
MATERIAL AND METHODS Sixty-one patients over the age of 40 having elective general surgical operations were admitted to the study. They were randomly allocated to a control (33 patients) and a test (28 patients) group. Patients in the test group received 0.2ml of sodium heparin (5,OOOiu), two hours before operation and then twelve-hourly for seven days using the method of Griffith and Boggs (1964) (14). The patients in the control group received 0.2ml of isotonic saline which was administered in the same manner. Collection of blood samples. 4.5ml of titrated blood was collected from each patient just before the pre-operative subcutaneous injection of heparin, then two, four and six hours subsequently on the day of operation. Blood was also collected just before and two hours after the morning injection on the first, third, and sixth postoperative days. The samples were centrifuged immediately at 2,500g for 30 minutes at 4"C, and 2ml of the supernatant platelet free plasma was removed and stored at -2O'C. All samples from each patient were assayed simultaneously. The AT-III assay was carried out on all samples except before heparin injection on the first, 3rd and 6th post operative day. It was carried out on M-Partigen immunodiffusion plates (Hoechst-Behring, Frankfurt, W. Germany) (15), which contain monospecific antiserum against the plasma protein-antithrombin III, in an agar-gel layer. The AT-III in the plasma forms a precipitin ring when in contact with the antibody in the agar. Antithrombin-III concentration is directly proportional to the square of the ring diameter. Protein standard plasma was used for calibration and the normal range obtained in our laboratory was 23-37mg%. The plasma heparin levels were estimated on all samples using a sensitive assay (16) based on the potentiating effect of heparin on the antifactor X . Reproducibility studies in our laboratory have shown that this assay coulda detect changes in heparin levels of O.O2u/ml of plasma.
RESULTS The mean levels of AT-III of the patients in both groups during the six hours after the first injection is shown in Figure 1. Figure 2 shows the mean levels of AT-III at 6 hours after injection on the day of operation and at 2 hours after injection on the first, third and sixth postoperative days. In the levels
control group, the maximum fall in the mean of plasma AT-III compared to pre-operation levels was reached on the third postoperative
Vol. 43, No. 6
AT-III HEPARIN & OPERATION
---
Control group II ~33
-
Test group
659
n-28
Compared to premop levels mpc 35,
---
Control group “=33
-
Test group
0.05
n=28
Compared to pre-op levels
@ D< 0.01
BP” 0.05 opt
31 Mean plasma At-Ill level mg/lOOml 29
0.01
I p< 0.05
35r
33
33
31
T
Mean plasma At-Ill level 29 mg/lOOml I’
25
21
23
25
21
23
\,:/
I
19 I
21I
I 0
J
0
2
4
6
I 1
3 Days
Hours
FIG.
FIG.
1
Mean + SD of AT-III in both groups during the six hours after first injection on the day of Paired t test was used. operation.
6
2
Mean + SD of AT-III at 6 hours after-the first injection of the day of operation, and at 2 hours after injection on the third and sixth postoperative day.
In only two of the 33 controls the level fell below the normal day (Fig 2). the maximum fall was at six hours after the first range. In the test group, injection, ie 2-3 hours after the end of operation (Fig 1). The level fell to This was the only time that a below the normal range in 7 of the 28 patients. significant difference was found when the means of the two groups were compared using a t-test.
jection AT-III
A wide variation in the plasma heparin levels There was no correlation between (Fig 3). levels at any time.
was found after each inthe heparin levels and the
660
AT-III
HEPARIN & OPERATION
Vol. 43, No. 6
.3a ,28 .26 .24 Plasma ‘22 hepark .20 units/ml .,6 % A4 .12 A0 ,08 .06 .04 .02 ,O 0246
02
Operation
Days
FIG. Median and 90 per and after 5,000 morning
02
1S.t
0
3rd
Zhours
6th
postoperative
3
cent tolerance level of heparin levels in the plasma units of subcutaneous heparin on the day of operation third and sixth postoperative day. of the first,
before and
DISCUSSION The results indicate that the plasma level of AT-III was decreased during and after operation in both the control and test groups, with a maximum fall in AT-III level was at six hours after operation. However, the decrease significantly greater in the test group than in the control group. This is the only time heparin has a marked effect on the consumption of AT-III. As this is the time of maximum risk of thrombosis, this finding might well explain why low dose heparin is effective as prophylaxis. The AT-III level during surgery has been studied by several igators; some reported a fall in the plasma AT-III level during (7-9,17); others found no significant changes (10,ll).
investoperation
Our results are in accord with previous reports in which sub-subcutaneous heparin given to patients with deep venous thrombosis and/or pulmonary embolism (18) and to patients with acute myocardial infarction (13)) induced a significant fall in the plasma level of AT-III. They support the hypothesis that AT-III is consumed during coagulation and its utilization is increased in the presence of heparin. Conflicting results have been shown by Kakkar (12) who reported increased AT-III values after subcutaneous heparin that was given to patients withextensive deep venous thrombosis using the same method for measuring plasma ATIII levels, as in our study. These findings (12) are contradicted by the findings of other workers (13,18) as well as our own data.
Vol. 43, No. 6
AT-III
661
HEPARIN & OPERATION
Other studies have investigated the effect of a continuous infusion of iv heparin (19,20) which has been shown to induce a decrease in the plasma levels of AT-III. The levels returned to normal 2-3 days after the discontinuation However, a single iv bolus of heparin did not seem to affect the of heparin. plasma AT-III level (20). It seems that heparin must be present in the blood for several hours in order to reduce AT-III concentrations. Studies on patients with congenital deficiencies of AT-III have shown that low levels of this inhibitor predispose to the development of thromboembolic episodes (21,22). It has been suggested that the low levels of AT-III during treatment with subcutaneous heparin may enhance the risk of developing thrombosis in the interval between the stopping of heparin and the return of AT-III levels to the normal range (20). This emphasizes the importance of adequate anticoagulation with oral agents prior to discontinuation of heparin therapy.
REFERENCES 1.
ROSENBERG, R.D. and DAMUS, P.S. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem, 248, 6490-6505, 1973
2.
GREIG, H.B.W. and NOTELOWITZ, M. levels. Lancet, I, 412-3, 1975
3.
VON KAULLA, E. and VON KAULLA, K.N. Oral contraceptives thrombin III activity. Lancet, 1, 36, 1970
4.
VON KAULLA, E. and VON KAULLA, K.N. Clin Pathol, 48, 69-80, 1967
5.
FAGERHOL, M.K., ABILDGAARD, U., BERGSJO, P. and JACOBSEN, J.H. Oral contraceptives and low antithrombin III concentration. Lancet, 1, 1175, 1970
6.
COLLINS, G.J., HEYMANN, R.L. and ZAJTCHUK, R. Hypercoagulability in patients with peripheral vascular disease. Am J Surg, 130, 2-6, 1975
7.
SAGAR, S., STAMATAKIS, J.D., THOMAS, D.P. and KAKKAR VV. Oral contraceptives, antithrombin III activity and postoperative deep vein thrombosis. Lancet, 1, 509-511, 1976
8.
STATHAKIS, N., PAPAYANNIS, A.G. and GARDIKAS, C.D. thrombin III concentration. Lancet, 1, 430, 1973
9.
COLLINS, G.J., BARBER, J.A., ZAJTCHUK, R., VANEK, D., MALOGNE, L.A. The effect of operative stress on the coagulation profile. Am J Surg, 133, 612-616, 1977
10.
HEDNER, U. and NILSSON, I.M. Antithrombin Thrombosis Res, 3, 631-641, 1973
11.
DONATI, M.B., VANDENBERGHE, K., MOLLA, A. DE ROO, M. and VERSTRAETE, Postoperative antithrombin III. Lancet, 1, 726-727, 1973
Natural oestrogens
Antithrombin
and antithrombin
III
and low anti-
III and diseases.
Postoperative
Am J
anti-
III in a clinical material.
M.
662
AT-III HEPARIN & OPERATION
Vol. 43, No. 6
12. KAKKAR, V. V., BENTLEY, P.G., SCLJLLY,M.F., MACGREGOR, I.R., JONES, N.A.G. and WEBB, P.J. Antithrombin III and heparin. Lancet, 1, 103-104, 1980 13.
GREEN P.J. Antithrombin III and subcutaneous heparin. 374-375, 1980
Lancet, 1,
14.
GRIFFITH, G.C. and BOGGS, R.P. Cardiol, 14, 39-46, 1964
15.
MANCINI, G., CARBONARA, A.O. and HEREMANS, J.F. Immunochemical quantitation of antigens by single radial immunodiffusion. Int J Immunochem, 2, 235-254, 1965
16.
DENSON, K.W. and BONNAR, J. The measurement of heparin. A method based on the.potentiation of antifactor X . Thromb Diath Haemorrh (Stuttg), a 30, 471-479, 1973
17.
SEYFER, A.E., SEABER, A.V., DOMBROSE, F.A. and URBANIAK, J.R. Coagulation changes in elective surgery and trauma. Ann Surg, 193, 210-213, 1981
18.
CONARD, J., LECOMPTE, T., HORELLOU, M.H., CAZENAVE, B. and SAMAMA, M. Antithrombin-III in patients treated with subcutaneous or intravenous heparin. Thrombosis Res, 22, 507-511, 1981
19.
MARCINIAK, E. and GOCKERMAN, J.P. Heparin-induced decrease in circulating antithrombin-III. Lancet, II, 581-584, 1977
20.
RAO, A.K, NIEWIAROWSKI, S., GUZZO, J. and DAY H.J. Antithrombin III levels during heparin therapy. Thrombosis Res, 24, 181-186, 1981
21.
EGBERG, 0. Inherited Antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh, 13, 516-530, 1965
22.
MARCINIAK, E., FARLEY, C. H. and DESIMONE, P.A. Familial thrombosis due to antithrombin-III deficiency. Blood, 43, 219-231, 1974
The clinical usage of heparin.
Am J