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Evaluation of the prethrombotic statein pregnancy and in women using oral contraceptives
ELSEVIER
Evaluation of the Prethrombotic State in Pregnancy and in Women Using Oral Contraceptives Amy B. Levine,* Jose Teppa,1- Becky McGough,1- and F. Susan CowchocklThe purpose of this study was to determine the effects of pregnancy and oral contraceptive use on in vivo coagulation by measurement of plasma levels of prothrombin fragment 1+2 and thrombin antithrombin III complex. Fifty-four women with uncomplicated singleton gestations, 34 women using oral contraceptives and 45 nonpregnant controls were the subjects of this study. Levels of prothrombin fragment 1+2 and thrombin-antithrombin III complex were measured using enzyme linked immunoassays. In uncomplicated pregnancies, levels of prothrombin fragment 1+2 and thrombin antithrombin III complex increased significantly with advancing gestational age. Women using oral contraceptives had significantly higher levels of prothrombin fragment 1+2 and thrombin antithrombin 111 complex compared to non-pregnant controls. The significant increase in levels of prothrombin fragment 1+2 and thrombin-antithrombin III complex throughout gestation and in women using oral contraceptives provided evidence of thrombin activation during these clinical conditions. CONTRACEPTION1996;53:255--257 KEY WORDS: prothrombin-fragment 1+2, thrombin antithrombin III complex, enzyme linked immunoassays
Introduction
p
regnancy and oral contraceptive use are two c o m m o n clinical conditions known to be associated with an increased risk of thrombosis. This association is believed to result from activation of the coagulation system, resulting in a prethrombotic state. The recent development of sensitive enzyme linked immunoassays that can detect and quantify a variety of complexes or peptide fragments generated as a re*Department of Obstetrics and Gynecology and 1-Departmentof Medicine, Jefferson Medical College, Philadelphia, PA, USA Name and address for correspondence: Amy B. Levine, MD., Jefferson Medical College, 834 Chestnut Street, Suite 400, Philadelphia, Pennsylvania 19107. Tel: (215) 955-7996; Fax: (215) 955-5041 Submitted for publication January 6, 1996 Revised February 13, 1996 Accepted for publication February 13, 1996
© 1996 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
sult of in vivo activation of the coagulation cascade may enable the biochemical identification of this prethrombotic state. Elevated levels of prothrombin fragm e n t 1+2 (Fl+2) and t h r o m b i n a n t i t h r o m b i n III complex (TAT) are markers of in vivo thrombin formation. 1'2 Specifically, thrombin results from the proteolysis of prothrombin by factor Xa into thrombin and the fragment F1+2. Subsequent to its generation, thrombin is inhibited by antithrombin III which forms an irreversible stable complex. The objective of this study was to determine the effects of pregnancy and oral contraceptive use on in vivo coagulation by measurement of plasma levels of p r o t h r o m b i n f r a g m e n t 1+2 and t h r o m b i n - a n t i thrombin III complex.
Materials and Methods Between June 1994 and July 1995, blood samples were obtained from w o m e n in the three study groups: pregnant, non-pregnant, and non-pregnant using oral contraceptives. Oral informed consent was obtained prior to venipuncture in accordance with institutional review board approval. Fifty-four women with uncomplicated singleton gestations who were obstetric patients of the faculty practice at Thomas Jefferson University Hospital were enrolled and evaluated in a cross-sectional manner. Forty-five non-pregnant women volunteered to serve as controls. Thirty-four women using oral contraceptives, either triphasic or monophasic low-dose estrogen combination products, also volunteers, were evaluated. Subjects with a history of thromboembolic disease, recent surgery, or use of medications (other than oral contraceptives) that might affect blood coagulation were excluded. Blood samples (5 ml) from the subjects were drawn into vacutainer tubes (Becton Dickinson, Rutherford, NJ) containing 0.105 mol/L buffered citrate. Plasma was separated from cellular elements by centrifugation at 3000 rpm for ten minutes and frozen in aliquots at 70°C until batch assays were performed. Assays were performed within four weeks of sample acquisition. ISSN 0010-7824/96/$15.00 PII S0010-7824(96)00057-1
256 Levine et al.
Contraception 1996;53:255-257
Commercially available enzyme linked immunoassays to measure the concentration of prothrombin fragment Fl+2 (Enzygost-Fl+2, Behring, Marburg, Germany) and thrombin-antithrombin III complex (Enzygost-TAT, Behring, Marburg, Germany) were used. A reference curve was prepared using the standards provided by the manufacturer with the ELISA kits. Samples were run in duplicate and the average was calculated. The reference ranges and assay sensitivities were provided by the manufacturer. The reference ranges for TAT and Fl+2 were 1.0 to 4.1 ng/ml and 0.4-1.1 nmol/L, respectively. T h e results were expressed as medians. The Kruskal-Wallis analysis of variance for censored data and the Mann-Whitney test were used for statistical analysis. P < 0.01 was considered significant.
Results The distribution of values for prothrombin fragment 1+2 and thrombin-antithrombin III complex in the non-pregnant control population is shown in Figures 1 and 2. Because the distribution was discontinuous, we calculated group medians and used nonparametric statistical testing for comparisons between the different groups. As shown in Table 1, median levels of both F 1+2 and TAT were significantly elevated in oral contraceptive users compared to non-pregnant controis. During pregnancy, levels of FI+2 and TAT increased with advancing gestational age (Table 2). Median values in the third trimester were significantly greater than those in the first and second trimesters. Values observed in the first trimester were comparable to those observed in the non-pregnant controls. 30
25
Number
20
with Value
15
35 30 25 Number with 20
Value
15 lO 5 o 1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16 17 18
(rig / ml)
Figure 2, Frequency distribution of plasma levels of thrombin-antithrombin III complex (TAT) in ng/ml in the non-pregnant control population.
Discussion The prevalence of thromboembolic events is clearly increased during pregnancy and the puerperium. The issue of whether modern (low-dose estrogen) oral contraceptive is associated with an increased risk of thrombosis is still controversial. With the availability of sensitive enzyme immunoassays that detect markers of thrombin generation, subtle degrees of activation of the coagulation cascade can be identified. In this study, two markers of thrombin generation, prothrombin fragment 1+2 and thrombin-antithrombin III complex, were evaluated in healthy non-pregnant women, oral contraceptive users, and healthy pregnant patients. Our observed values for these markers in the nonpregnant women were within the expected range as previously reported.l'3'4 However, for both markers, a substantial number of controls had markedly elevated values (Figures 1 and 2). This finding has been observed by others. 3'5 These abnormal values may be the result of traumatic venipuncture or may represent other as yet u n i d e n t i f i e d factors that influence plasma levels. This raises the concern that these assays may have suboptimal specificity. We found significantly elevated levels of both F1+2 and TAT in oral contraceptive users compared with controls. Three prior investigations have evaluated these markers in oral contraceptive users, s-7 Jespersen et al.
Table 1, Median levels of Fl+2 and TAT in non-pregnant controls and oral contraceptive users
5
1
2
3
4
5
(nmol
6
7
8
9
10
11
N
Fl+2 (nmol/L)
TAT (ng/ml)
45 34
1.3 1.9"
1.7 2.3t
/ L)
Figure 1, Frequency distribution of plasma levels of pro-
thrombin fragment 1+2 (Fl+2) in nmol/L in the nonpregnant control population.
Controls OC users
O.C. = Oral contraceptives.
*p = 0 . 0 0 5 ; t p = 0 . 0 0 8 .
Contraception 1996;53:255-257
Prethrombotic States in Women
Table 2. Median levels of Fl+2 and TAT in pregnancy
N
(nmol/L)
TAT (ng/ml)
16 18 20
1.8 2.1 3.4*
2.0 3.5 7.2t
F1+2 First trimester Second trimester Third trimester *p = 0.001; fp = 0.0001.
evaluated 19 w o m e n before and during treatment with low-dose estrogen oral contraceptives and found no difference in TAT levels. Saleh et al. compared 56 contraceptive users to 47 controls and found no significant differences in either TAT or Fl+2 levels. On the contrary, Gram et al. found significantly elevated levels of TAT in 28 contraceptive users compared with 30 controls, a result consistent with our finding. Further i n v e s t i g a t i o n is n e c e s s a r y to d e t e r m i n e whether currently used oral contraceptives activate the clotting mechanism. Our observation of increasing levels of both Fl+2 and TAT as pregnancy advances corroborate those of previous investigations.8-1~ de Boer et al. s and Cadroy et al. 9 evaluated w o m e n with uncomplicated pregnancies in a cross-sectional manner and observed a significant increase in plasma TAT levels with advancing gestational age, though their results were not analyzed by trimester. In another cross-sectional study of 83 normal pregnant women, Reinthaller et a l ) ° found that mean TAT concentrations increased significantly in the second (5.0 ng/ml) and third (4.8 ng/ml) trimesters compared with values in the first trimester (3.1 ng/ml). Bremme et a l ) evaluated 26 uncomplicated pregnant w o m e n longitudinally and found that up to 15 weeks gestation only 11 of 22 w o m e n had TAT levels greater than the reference range with a mean value of 3.1 ng/ml. After 24 weeks gestation, all w o m e n had significantly elevated levels, with mean values of 5.9 ng/ml at 24 weeks and 7.1 ng/ml at 35 weeks gestation. Our study also demonstrates a significant increase in TAT with gestational age. Only one previously cited published article has evaluated F1+2 in normal pregnancies and found, as we did, that levels of F1+2 increased significantly with gestational age. 9 The results from this study provide further evidence for the prethrombotic state of pregnancy and suggest that oral contraceptive use m a y also produce a prethrombotic state. The clinical utility of measure-
257
m e n t of these markers in various conditions associated with an increased risk of thrombosis is unclear because the prevalence of elevated levels in our normal population was high.
Acknowledgment The authors would like to acknowledge the manufacturer, Behring, Marburg, Germany, for their generous contribution of the enzyme linked immunoassay kits.
References 1. Bauer KA, Rosenberg RD. The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of hemostatic system activation. Blood 1987;70:343-50. 2. Saleh AA, Dorey LG, Dombrowski MP, et al. Thrombosis and hormone replacement therapy in postmenopausal women. Am J Obstet Gynecol 1993;169:1554-7. 3. Hursting MJ, Stead AG, Crout FV, Horvath BZ, Moore BM. Effects of age, race, sex and smoking on prothrombin fragment 1+2 in a healthy population. Clin Chem 1993;39:683-6. 4. Pelzer H, Schwarz A, Heimburger N. Determination of human thrombin-antithrombin III complex in plasma with an enzyme linked immunoassay. Thromb Haemost 1988~59:101-6. 5. Jespersen J, Petersen KR, Skovby SO. Effects of newer oral contraceptives on the inhibition of coagulation and fibrinolysis in relation to dosage and type of steroid. Am J Obstet Gynecol 1990;163:396-403. 6. Saleh AA, Brochbank N, Dorey LG, Ozawa T, Dombrowksi MP, Bottoms SF, et al. TAT complexes and prothrombin fragment 1+2 in oral contraceptive users. Thromb Res 1994;73:137-42. 7. Gram J, Munkvad S, Jespersen J. Enhanced generation and resolution of fibrin in women above the age of 30 years using oral contraceptives low in estrogen. Am J Obstet Gynecol 1990;163:438-42. 8. de Boer K, TenCate JW, Sturk A, Borm JJ, Treffers PE. Enhanced thrombin generation in normal and hypertensive pregnancy. Am J Obstet Gynecol 1989;160:95100. 9. Cadroy Y, Grandjean H, Pichon J, et al. Evaluation of six markers of haemostatic system in normal pregnancy and pregnancy complicated by hypertension or pre-eclampsia. Br J Obstet Gynecol 1993;100:416-20. 10. Reinthaller A, Mursch-Edlmayr G, Tatra G. Thrombinantithrombin III complex levels in normal pregnacy with hypertensive disorders and after delivery. Br J Obstet Gynecol 1990;97:506-10. 11. Bremme K, Ostlund E, Almqvist I, Heinonen K, Blomback M. Enhanced thrombin generation and fibrinolytic activity in normal pregnancy and the puerperium. Obstet Gynecol 1992;80:132-7.
Evaluation of the Prethrombotic State in Pregnancy and in Women Using Oral Contraceptives Amy B. Levine,* Jose Teppa,1- Becky McGough,1- and F. Susan CowchocklThe purpose of this study was to determine the effects of pregnancy and oral contraceptive use on in vivo coagulation by measurement of plasma levels of prothrombin fragment 1+2 and thrombin antithrombin III complex. Fifty-four women with uncomplicated singleton gestations, 34 women using oral contraceptives and 45 nonpregnant controls were the subjects of this study. Levels of prothrombin fragment 1+2 and thrombin-antithrombin III complex were measured using enzyme linked immunoassays. In uncomplicated pregnancies, levels of prothrombin fragment 1+2 and thrombin antithrombin III complex increased significantly with advancing gestational age. Women using oral contraceptives had significantly higher levels of prothrombin fragment 1+2 and thrombin antithrombin 111 complex compared to non-pregnant controls. The significant increase in levels of prothrombin fragment 1+2 and thrombin-antithrombin III complex throughout gestation and in women using oral contraceptives provided evidence of thrombin activation during these clinical conditions. CONTRACEPTION1996;53:255--257 KEY WORDS: prothrombin-fragment 1+2, thrombin antithrombin III complex, enzyme linked immunoassays
Introduction
p
regnancy and oral contraceptive use are two c o m m o n clinical conditions known to be associated with an increased risk of thrombosis. This association is believed to result from activation of the coagulation system, resulting in a prethrombotic state. The recent development of sensitive enzyme linked immunoassays that can detect and quantify a variety of complexes or peptide fragments generated as a re*Department of Obstetrics and Gynecology and 1-Departmentof Medicine, Jefferson Medical College, Philadelphia, PA, USA Name and address for correspondence: Amy B. Levine, MD., Jefferson Medical College, 834 Chestnut Street, Suite 400, Philadelphia, Pennsylvania 19107. Tel: (215) 955-7996; Fax: (215) 955-5041 Submitted for publication January 6, 1996 Revised February 13, 1996 Accepted for publication February 13, 1996
© 1996 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
sult of in vivo activation of the coagulation cascade may enable the biochemical identification of this prethrombotic state. Elevated levels of prothrombin fragm e n t 1+2 (Fl+2) and t h r o m b i n a n t i t h r o m b i n III complex (TAT) are markers of in vivo thrombin formation. 1'2 Specifically, thrombin results from the proteolysis of prothrombin by factor Xa into thrombin and the fragment F1+2. Subsequent to its generation, thrombin is inhibited by antithrombin III which forms an irreversible stable complex. The objective of this study was to determine the effects of pregnancy and oral contraceptive use on in vivo coagulation by measurement of plasma levels of p r o t h r o m b i n f r a g m e n t 1+2 and t h r o m b i n - a n t i thrombin III complex.
Materials and Methods Between June 1994 and July 1995, blood samples were obtained from w o m e n in the three study groups: pregnant, non-pregnant, and non-pregnant using oral contraceptives. Oral informed consent was obtained prior to venipuncture in accordance with institutional review board approval. Fifty-four women with uncomplicated singleton gestations who were obstetric patients of the faculty practice at Thomas Jefferson University Hospital were enrolled and evaluated in a cross-sectional manner. Forty-five non-pregnant women volunteered to serve as controls. Thirty-four women using oral contraceptives, either triphasic or monophasic low-dose estrogen combination products, also volunteers, were evaluated. Subjects with a history of thromboembolic disease, recent surgery, or use of medications (other than oral contraceptives) that might affect blood coagulation were excluded. Blood samples (5 ml) from the subjects were drawn into vacutainer tubes (Becton Dickinson, Rutherford, NJ) containing 0.105 mol/L buffered citrate. Plasma was separated from cellular elements by centrifugation at 3000 rpm for ten minutes and frozen in aliquots at 70°C until batch assays were performed. Assays were performed within four weeks of sample acquisition. ISSN 0010-7824/96/$15.00 PII S0010-7824(96)00057-1
256 Levine et al.
Contraception 1996;53:255-257
Commercially available enzyme linked immunoassays to measure the concentration of prothrombin fragment Fl+2 (Enzygost-Fl+2, Behring, Marburg, Germany) and thrombin-antithrombin III complex (Enzygost-TAT, Behring, Marburg, Germany) were used. A reference curve was prepared using the standards provided by the manufacturer with the ELISA kits. Samples were run in duplicate and the average was calculated. The reference ranges and assay sensitivities were provided by the manufacturer. The reference ranges for TAT and Fl+2 were 1.0 to 4.1 ng/ml and 0.4-1.1 nmol/L, respectively. T h e results were expressed as medians. The Kruskal-Wallis analysis of variance for censored data and the Mann-Whitney test were used for statistical analysis. P < 0.01 was considered significant.
Results The distribution of values for prothrombin fragment 1+2 and thrombin-antithrombin III complex in the non-pregnant control population is shown in Figures 1 and 2. Because the distribution was discontinuous, we calculated group medians and used nonparametric statistical testing for comparisons between the different groups. As shown in Table 1, median levels of both F 1+2 and TAT were significantly elevated in oral contraceptive users compared to non-pregnant controis. During pregnancy, levels of FI+2 and TAT increased with advancing gestational age (Table 2). Median values in the third trimester were significantly greater than those in the first and second trimesters. Values observed in the first trimester were comparable to those observed in the non-pregnant controls. 30
25
Number
20
with Value
15
35 30 25 Number with 20
Value
15 lO 5 o 1
2
3
4
5
6
7
8
9
10 11 12
13
14
15
16 17 18
(rig / ml)
Figure 2, Frequency distribution of plasma levels of thrombin-antithrombin III complex (TAT) in ng/ml in the non-pregnant control population.
Discussion The prevalence of thromboembolic events is clearly increased during pregnancy and the puerperium. The issue of whether modern (low-dose estrogen) oral contraceptive is associated with an increased risk of thrombosis is still controversial. With the availability of sensitive enzyme immunoassays that detect markers of thrombin generation, subtle degrees of activation of the coagulation cascade can be identified. In this study, two markers of thrombin generation, prothrombin fragment 1+2 and thrombin-antithrombin III complex, were evaluated in healthy non-pregnant women, oral contraceptive users, and healthy pregnant patients. Our observed values for these markers in the nonpregnant women were within the expected range as previously reported.l'3'4 However, for both markers, a substantial number of controls had markedly elevated values (Figures 1 and 2). This finding has been observed by others. 3'5 These abnormal values may be the result of traumatic venipuncture or may represent other as yet u n i d e n t i f i e d factors that influence plasma levels. This raises the concern that these assays may have suboptimal specificity. We found significantly elevated levels of both F1+2 and TAT in oral contraceptive users compared with controls. Three prior investigations have evaluated these markers in oral contraceptive users, s-7 Jespersen et al.
Table 1, Median levels of Fl+2 and TAT in non-pregnant controls and oral contraceptive users
5
1
2
3
4
5
(nmol
6
7
8
9
10
11
N
Fl+2 (nmol/L)
TAT (ng/ml)
45 34
1.3 1.9"
1.7 2.3t
/ L)
Figure 1, Frequency distribution of plasma levels of pro-
thrombin fragment 1+2 (Fl+2) in nmol/L in the nonpregnant control population.
Controls OC users
O.C. = Oral contraceptives.
*p = 0 . 0 0 5 ; t p = 0 . 0 0 8 .
Contraception 1996;53:255-257
Prethrombotic States in Women
Table 2. Median levels of Fl+2 and TAT in pregnancy
N
(nmol/L)
TAT (ng/ml)
16 18 20
1.8 2.1 3.4*
2.0 3.5 7.2t
F1+2 First trimester Second trimester Third trimester *p = 0.001; fp = 0.0001.
evaluated 19 w o m e n before and during treatment with low-dose estrogen oral contraceptives and found no difference in TAT levels. Saleh et al. compared 56 contraceptive users to 47 controls and found no significant differences in either TAT or Fl+2 levels. On the contrary, Gram et al. found significantly elevated levels of TAT in 28 contraceptive users compared with 30 controls, a result consistent with our finding. Further i n v e s t i g a t i o n is n e c e s s a r y to d e t e r m i n e whether currently used oral contraceptives activate the clotting mechanism. Our observation of increasing levels of both Fl+2 and TAT as pregnancy advances corroborate those of previous investigations.8-1~ de Boer et al. s and Cadroy et al. 9 evaluated w o m e n with uncomplicated pregnancies in a cross-sectional manner and observed a significant increase in plasma TAT levels with advancing gestational age, though their results were not analyzed by trimester. In another cross-sectional study of 83 normal pregnant women, Reinthaller et a l ) ° found that mean TAT concentrations increased significantly in the second (5.0 ng/ml) and third (4.8 ng/ml) trimesters compared with values in the first trimester (3.1 ng/ml). Bremme et a l ) evaluated 26 uncomplicated pregnant w o m e n longitudinally and found that up to 15 weeks gestation only 11 of 22 w o m e n had TAT levels greater than the reference range with a mean value of 3.1 ng/ml. After 24 weeks gestation, all w o m e n had significantly elevated levels, with mean values of 5.9 ng/ml at 24 weeks and 7.1 ng/ml at 35 weeks gestation. Our study also demonstrates a significant increase in TAT with gestational age. Only one previously cited published article has evaluated F1+2 in normal pregnancies and found, as we did, that levels of F1+2 increased significantly with gestational age. 9 The results from this study provide further evidence for the prethrombotic state of pregnancy and suggest that oral contraceptive use m a y also produce a prethrombotic state. The clinical utility of measure-
257
m e n t of these markers in various conditions associated with an increased risk of thrombosis is unclear because the prevalence of elevated levels in our normal population was high.
Acknowledgment The authors would like to acknowledge the manufacturer, Behring, Marburg, Germany, for their generous contribution of the enzyme linked immunoassay kits.
References 1. Bauer KA, Rosenberg RD. The pathophysiology of the prethrombotic state in humans: insights gained from studies using markers of hemostatic system activation. Blood 1987;70:343-50. 2. Saleh AA, Dorey LG, Dombrowski MP, et al. Thrombosis and hormone replacement therapy in postmenopausal women. Am J Obstet Gynecol 1993;169:1554-7. 3. Hursting MJ, Stead AG, Crout FV, Horvath BZ, Moore BM. Effects of age, race, sex and smoking on prothrombin fragment 1+2 in a healthy population. Clin Chem 1993;39:683-6. 4. Pelzer H, Schwarz A, Heimburger N. Determination of human thrombin-antithrombin III complex in plasma with an enzyme linked immunoassay. Thromb Haemost 1988~59:101-6. 5. Jespersen J, Petersen KR, Skovby SO. Effects of newer oral contraceptives on the inhibition of coagulation and fibrinolysis in relation to dosage and type of steroid. Am J Obstet Gynecol 1990;163:396-403. 6. Saleh AA, Brochbank N, Dorey LG, Ozawa T, Dombrowksi MP, Bottoms SF, et al. TAT complexes and prothrombin fragment 1+2 in oral contraceptive users. Thromb Res 1994;73:137-42. 7. Gram J, Munkvad S, Jespersen J. Enhanced generation and resolution of fibrin in women above the age of 30 years using oral contraceptives low in estrogen. Am J Obstet Gynecol 1990;163:438-42. 8. de Boer K, TenCate JW, Sturk A, Borm JJ, Treffers PE. Enhanced thrombin generation in normal and hypertensive pregnancy. Am J Obstet Gynecol 1989;160:95100. 9. Cadroy Y, Grandjean H, Pichon J, et al. Evaluation of six markers of haemostatic system in normal pregnancy and pregnancy complicated by hypertension or pre-eclampsia. Br J Obstet Gynecol 1993;100:416-20. 10. Reinthaller A, Mursch-Edlmayr G, Tatra G. Thrombinantithrombin III complex levels in normal pregnacy with hypertensive disorders and after delivery. Br J Obstet Gynecol 1990;97:506-10. 11. Bremme K, Ostlund E, Almqvist I, Heinonen K, Blomback M. Enhanced thrombin generation and fibrinolytic activity in normal pregnancy and the puerperium. Obstet Gynecol 1992;80:132-7.