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Oral contraceptives reduce total protein S, but not free protein S.
THROMBOSIS RESEARCH 45; 109-114, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
BRIEF
COMMUNICATION
ORAL CONTRACEPTIVES REDUCE TOTAL PROTEIN S, BUT NOT FREE PROTEIN S.
I.A. Huisveld, J.E.H. Hospers, J.C.M. Meijers, A.E. Starkenburg, W.B.M. Erich and B.N. Bouma. Departments of Physiology and Haematology, State University of Utrecht, 3521 GG Utrecht, The Netherlands. (Received 10.8.1986; Accepted in revised form 5.9.1986 by Editor P.M. Mannucci)
INTRODUCTION
Despite a reduction in the oestrogen content, the use of oral contraceptive agents (OCA) is still associated with an increased risk for thromboembolic complications (1). Among the many studies that have been performed to elucidate the mechanism responsible for the enhanced morbidity are those dealing with the haemostatic mechanism (2, 3). OCA have been shown to induce changes in a number of coagulation and fibrinolytic factors (2, 3, 4). The extent to which these changes may be of causal significance in thrombotic disease is still a matter of debate. A clear effect of OCA has been observed on plasma levels of vitamin K-dependent coagulation factors (3, 5). Recently two new vitamin K-dependent proteins, i.e. protein C and protein S, involved in haemostasis have been identified (6, 7).Protein C (PC) is a serine protease which has potent anticoagulant properties. In its activated form (APC), PC has been shown to inactivate the coagulation factors Va and VIIIa, thus inhibiting the formation of thrombin (8, 9). The fact that APC enhances blood clot lysis both in vivo (10) and in vitro (11, 12) suggests an additional profibrinolytic role for this protein. Protein S (PS) circulates in plasma as a free protein and in reversible complex with C4b-binding protein (C4b-bp), a regulatory protein of the complement system (13). Free protein (PS-free) functions as an obligatory non-enzymatic cofactor for the effects of APC (9, 14). Recent studies have demonstrated that patients deficient in either PC (15) or PS (16) are subject to recurrent venous thromboembolic disease. In the present study the effects of low-dose oestrogen contraceptive medication on FII, PC,PS and C4b-bp are assessed in healthy women.In order to minimize the reported influence of variables such as age , body composition , nutritional habits and physical conditioning (2, 5, 17) we performed our study in a homogeneous group of equally well-trained competitive rowers.
Key words: Oral contraceptives - blood coagulation - protein S - protein C C4b-binding protein. 109
110
ORAL CONTRACEPTIVES & PROTEIN S
Vol. 45, No. 1
SUBJECTS AND METHODS
Subjects: 20 highly trained female athletes taking part in national and international competitive rowing events participated in this study. Informed consent was obtained after an explanation of the study s objectives. Subjects were divided into two groups. The group of users had been using OCA for more than a year. 8 of these women used OCA containing 0.03 mg ethinyloestradiol and 0.15 mg levonorgestrel, 2 others used a combination of 0.05 mg ethinyloestradiol with 1 mg lynestrenol. The 10 athletes serving as controls had not been on oral contraceptive medication for more than a year and had regular menstrual cycles. All participants trained for 3 hrs daily and refrained from smoking. Physical conditioning of the subjects, expressed as maximum oxygen uptake (VO by means of an increasing work load test on a bicyc 1 e max), was determined ergometer as described previously (4).
Body composition: Percentage bodyfat measured with an underwater weighing culated (4)
was derived from body density which was technique. Lean body mass was then cal-
Blood sampling and preparation of plasma: Blood collection was performed in the morning hours (to exclude diurnal variations) during the follicular phase of the menstrual cycle and between days 1-14 of the hormone-induced cycle. All blood samples were drawn in upright position from the antecubital vein by clean venipuncture, using a 1.2 mm bore needle. The first 2 ml were discarded and the next 9 ml were collected in a plastic tube containing 1 ml of 3.8% trisodium citrate-2-hydrate for the assay of the haemostatic factors. Citrated blood was immediately centrifuged (4 x 103g) for 10 min at room temperature. Platelet poor plasma was divided into small aliquots, snap frozen and stored at -7O'C to be assayed.
Assay of coagulation factors: Plasma antigen levels of FII, PC, total PS, PSfree and C4b-bp were determined by Laurel1 rocket immunoelectrophoresis. Gels for the Laurel1 plates contained 0.9% agarose (Seakem, Rockland ME, USA), 8 mmol/l EDTA(Sigma,St Louis MO, USA) and 2% PEG 8000 (Sigma, St Louis MO, USA) in 0.08 mol/l Tris/0.025 mol/l Tricine buffer pH 8.6. The same buffer with 8 mmol/l EDTA was employed for electrophoresis. To the gels 0.75% anti-F11 (Clotimmun-Prothrombin, Behringwerke, Marburg, FRG), 0.7% anti-PS (see below) and 1% anti-C4b-bp (Behring Diagnostics, La Jolla Ca, USA) were added for the assays of FII, PS and C4b-bp respectively. Staining of the plates was performed with Coomassie Brilliant Blue R 250 (Merck, Darmstadt, FRG). PC antigen levels were measured using a commercially available test kit (Assera.plate protein C, Diagnostica Stago, Asnieres, France), according to the instructions of the manufacturer. Each Laurel1 plate was standardized with four dilutions (25, 50, 75, 100%) of a normal plasma pool (n=20). Two dilutions of each plasma sample were analyzed and the antigen levels were calculated and expressed relative to the normal pool which was defined as 100%. For the separation of the complexed form of PS (PS-C4b-bp) and the free form of PS (PS-free) a technique was used based on the method recently described by Comp et al (18). Addition of PEG 8000 to plasma in a final concentration of 4.2% resulted in the precipitation of PS-Cbb-bp, while PS-free remained in the supernatant as was confirmed by crossed immunoelectrophoresis (18). Laurel1 plates containing 0.7% anti-PS were used to analize the supernatant. A standard
Vol. 45, No. 1
ORAL CONTRACEPTIVES & PROTEIN S
111
curve from normal pooled plasma that had been treated in the same way was included in each plate. Electrophoresis for total PS (free and bound PS) and PS-free was conducted at 25 'C with 5V/cm for 15-18 hrs. Under these conditions a single peak for total PS was observed. Anti-PS was prepared in rabbits using purified PS (19). The antiserum gave one precipitation line when tested in double immunodiffusion against normal plasma. This line showed a reaction of identity with the precipitation line obtained against purified PS. Anti-PS inhibited the cofactor activity of PS in normal plasma (20).
Statistical analysis: Means and standard deviations were calculated with the usual methods. Date were compared by Student's t-test or by Mann-Whitney test as appropriate. Differences were considered statistically significant at P
RESULTS
The effect of OCA on PC and PS was studied in two groups of highly trained athletes. The.groups were very well matched, no significant differences between users and controls were observed in age (23.7 + 1.8 and 23.4 5 2.2 year), weight (69.3 + 4.8 and 70.8 + 6.3 kg), body fat (24.7 + 4.3 and 23.3 2 2.5%) and physical conditioning (3.84 2 0.44 and 4.18 + 0.32 irO;-max l/min). In the group of OCA-users a significant lower PS antigen level (P
TABLE I Antigen levels of Plasma Proteins
Controls (n=lO> Factor II Protein C Protein S C4-b-binding protein Protein S-free
112.7 + 108.4 T 111.7 T 96.4 + 104.6 T
17.2 20.4 21.8 13.8 15.2
Users (n=lO) 113.2 + 109.7 T 84.5 + 81.5 T 94.3 z
13.6 33.0 19.0 * 14.0 * 18.0
Values expressed as % of normal pooled plasma. Means + SD are presented. * Significant differences between the groups P
In plasma PS circulates partly in a complex with C4b-bp. The level of C4b-bp therefore, affects the level of PS-free. In the group of users a significantly decreased level of C4b-bp (P
112
ORAL CONTRACEPTIVES & PROTEIN S
Vol.
45, No. 1
DISCUSSION
Despite a gradual reduction in oestrogens and progestagens, the use of OCA is still associated with an enhanced risk for thromboembolism in a number of women (1). It has been established that the observed venous thromboembolic complications were strongly related to the oestrogen dose of OCA (2). A comparable association has been reported between the oestrogen content and the magnitude of the changes observed in the levels of vitamin K-dependent clotting factors (3, 4, 5). The present investigation was undertaken following the recent publication of studies reporting a clear relation between thromboembolic disease and depressed levels of either PC (15) or PS (16). The effect of low-dose OCA on FII, PC, PS and C4b-bp was studied in healthy equally well-conditioned women. The most striking observations in our study were the reduced levels of PS and C4b-bp in relation to OCA medication. In view of the association between low PS plasma levels and venous thrombosis, our findings might well explain, at first sight, the enhanced incidence of thromboembolic complications reported in users of OCA. The interpretation of changes in (total) PS is, however, complicated by the fact that only a fraction of the protein (PS-free) is active as a cofactor for the anticoagulant effect of activated PC (14). The major part of PS bound to C4b-bp, an inhibitory protein of the complement system, is functionally inactive (18). C4b-bp has a potential regulatory function with respect to the activity of PS, since changes in the plasma levels of this protein determine the distribution between bound and free forms of PS. In our study the OCArelated reduction in total PS was paralleled by a reduction in C4b-bp. The compensatory changes in C4b-bp resulted in normal PS-free plasma levels that were fully comparable to those observed in the controls. It is quite conceivable that subjects who fail to maintain the optimal equilibrium between PS and Clrb-bp plasma levels might be prone to an enhanced thrombogenecity. Interesting in this respect are the reported shifts from free to bound PS in pregnancy, nephrotic syndrome and lupus erythematosus, conditions that have been associated with thromboembolic complications (21).The close relation between the complement and the coagulation system is stressed by earlier studies (4,22) that reported OCA-induced reductions in Cl-inactivator, another plasma protein involved in both systems. OCA-related increases in the plasma levels of FII and PC, as reported previously (3, 5), were not observed in the group of users. The fact that the subjects in our study were well-conditioned and used OCA with a low oestrogen content might explain the discrepancy. From the results of our study we conclude that a reduction in the plasma level of total PS is not necessarily associated with a lower level of free, functionin the group of users ally active PS. The normal levels of PC and PS-free suggest that OCA medication has no adverse effect on the protein C system in healthy individuals. The OCA-related enhanced fibrinolytic potential observed in a comparable group of athletes (4) could support this conclusion.
REFERENCES
1. BuTTIGER,L.E., thromboembolic 101,198O.
BOMAN,G., EKLUND,G. and WESTERHOLM,B. Oral contraceptives and disease: effect of lowering estrogen content. Lancet 1,1097-
2. MEADE,T.W. Oral contraceptives, Gynaecols,758-61,1982.
clotting factors and thrombosis.Am
J Obstet
Vol. 45, No. 1
ORAL CONTRACEPTIVES & PROTEIN S
113
3.
MAMMEN,E.F. Oral contraceptives and blood coagulation:a critical review. Am J Obstet GynecolE,781-790,1982.
4.
HUISVELD,I.A., KLUFT,C., HOSPERS,J.E.H., BERNINK,M.J.E., ERICH,W.B.M. and BOUMA,B.N. Effect of exercise and oral contraceptive agents on fibrinolytic potential in trained females. J Appl Physiol REEP x,906-13,1984.
5.
MEADE,T.W., STIRLING,Y.,WILKES,H. and MANNUCC1,P.M. Effects of oral contraceptives and obesity on protein C antigen. Thromb Haemost =,198-9,1985.
6.
STENFLO,J. A new vitamin K-dependent protein: purification from bovine plasma and preliminary characterization. J Biol Chem =,355-63,1976.
7.
DI SCIPIO,R.G., HERMODSON,M.A., YATES,S.G. and DAVIE,E.W. A comparison of human prothrombin, factor IX, factor X and protein S. Biochemistry *,698706,1977.
8.
MARLAR,R.A., KLEISS,A.J. and GRIFFIN,J.H. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 2,1067-72,1982.
9.
CLOUSE,L.H. and COMP,P.C. The regulation of haemostasis: the protein C system. N Engl J Med x,1298-304,1986.
10.
COMP,P.C. and ESMON,C.T. Generation of fibrinolytic activity by infusion of activated protein C into dogs. J Clin Invest E,1221-8,198l.
11.
TAYLOR,F.B. and LOCKHART,M.S. Whole blood clot lysis: in vitro modulation by activated protein C. Thromb Res x,639-49,1984.
12.
DE FOUW,N.J., HAVERKATE,F., BERTINA,R.M., KOOPMAN,J.,VAN WIJNGAARDEN,A. and VAN HINSBERGH,V.W.M. The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro. -Blood 67,118992,1986.
13.
DAHLB#CK,B. and STENFLO,J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Nat1 Acad Sci USAz,2512-6,198l.
14.
WALKER,F.J. Regulation of activated protein C by a new protein: a possible function for bovine protein S. J Biol Chem 255, 5521-4,198O.
15.
GRIFFIN,J.H., EVATTS,B., ZIMMERMAN,T.S., KLEISS,A.J. and WIDEMAN,C. Deficiency of protein C in congenital thrombotic disease.J Clin Invest 68,1370-3,1982.
16.
SCHWARZ,H.P., FISCHER,M., HOPMEIER,P., BATARD,M.A. and GRIFFIN,J.H. Plasma protein S deficiency in familial thrombotic disease. -Blood 311,1525-8,1984.
17.
WALLEWIS,J.L., BARTON,T., STEAD,N.W., WILLIAMS,R.S., LOGUE,E.E., LACE,A.G.and PIZZ0,S.V. Physical conditioning augments the fibrinolytic response to venous occlusion in healthy adults. N Engl J Med =,98791,198O.
18.
COMP,P.C., DORAY,D., PATTON,D.and ESMON,C.T. An abnormal plasma distribution of protein S occurs in functional protein S deficiency.Blood 67,504-8,1986.
114
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Vol. 45, No. 1
and MEIJERS,J.C.M. Immunochemical 19. BOIJMA,B.N., VLOOSWIJK,R.A.A. ation of human protein S. Thromb Haemost z,83,1985. VLOOSWIJK,R.A.A., KOEDAM,J.A., 20. BOUMA,B.N., Characterization of human MEIJERS,J.C.M. 6(supp1),135,1986.
characteriz-
STARKENBURG,A.E. protein S. Thromb
and Res
21. COMP,P.C., VIGANO,S., D'ANGELO,A., THURNEAU,G., KAUFMAN,C. and ESMON,C.T. Acquired protein S deficiency occurs in pregnancy, the nephrotic syndrome and acute systemic lupus erythematosus. Blood 66(supp11),348a,1985. 22. GORDON,E.M., RATNOFF,O.D., SAITO,H., DONALDSON,V.H., PENSKY,J. and JONES,P.K. Rapid fibrinolysis, augmented Hageman factor (factor XII) titers and decreased Cl-esterase inhibitor titers in women taking contraceptives. J Lab Clin Med =,762-9,198O.
BRIEF
COMMUNICATION
ORAL CONTRACEPTIVES REDUCE TOTAL PROTEIN S, BUT NOT FREE PROTEIN S.
I.A. Huisveld, J.E.H. Hospers, J.C.M. Meijers, A.E. Starkenburg, W.B.M. Erich and B.N. Bouma. Departments of Physiology and Haematology, State University of Utrecht, 3521 GG Utrecht, The Netherlands. (Received 10.8.1986; Accepted in revised form 5.9.1986 by Editor P.M. Mannucci)
INTRODUCTION
Despite a reduction in the oestrogen content, the use of oral contraceptive agents (OCA) is still associated with an increased risk for thromboembolic complications (1). Among the many studies that have been performed to elucidate the mechanism responsible for the enhanced morbidity are those dealing with the haemostatic mechanism (2, 3). OCA have been shown to induce changes in a number of coagulation and fibrinolytic factors (2, 3, 4). The extent to which these changes may be of causal significance in thrombotic disease is still a matter of debate. A clear effect of OCA has been observed on plasma levels of vitamin K-dependent coagulation factors (3, 5). Recently two new vitamin K-dependent proteins, i.e. protein C and protein S, involved in haemostasis have been identified (6, 7).Protein C (PC) is a serine protease which has potent anticoagulant properties. In its activated form (APC), PC has been shown to inactivate the coagulation factors Va and VIIIa, thus inhibiting the formation of thrombin (8, 9). The fact that APC enhances blood clot lysis both in vivo (10) and in vitro (11, 12) suggests an additional profibrinolytic role for this protein. Protein S (PS) circulates in plasma as a free protein and in reversible complex with C4b-binding protein (C4b-bp), a regulatory protein of the complement system (13). Free protein (PS-free) functions as an obligatory non-enzymatic cofactor for the effects of APC (9, 14). Recent studies have demonstrated that patients deficient in either PC (15) or PS (16) are subject to recurrent venous thromboembolic disease. In the present study the effects of low-dose oestrogen contraceptive medication on FII, PC,PS and C4b-bp are assessed in healthy women.In order to minimize the reported influence of variables such as age , body composition , nutritional habits and physical conditioning (2, 5, 17) we performed our study in a homogeneous group of equally well-trained competitive rowers.
Key words: Oral contraceptives - blood coagulation - protein S - protein C C4b-binding protein. 109
110
ORAL CONTRACEPTIVES & PROTEIN S
Vol. 45, No. 1
SUBJECTS AND METHODS
Subjects: 20 highly trained female athletes taking part in national and international competitive rowing events participated in this study. Informed consent was obtained after an explanation of the study s objectives. Subjects were divided into two groups. The group of users had been using OCA for more than a year. 8 of these women used OCA containing 0.03 mg ethinyloestradiol and 0.15 mg levonorgestrel, 2 others used a combination of 0.05 mg ethinyloestradiol with 1 mg lynestrenol. The 10 athletes serving as controls had not been on oral contraceptive medication for more than a year and had regular menstrual cycles. All participants trained for 3 hrs daily and refrained from smoking. Physical conditioning of the subjects, expressed as maximum oxygen uptake (VO by means of an increasing work load test on a bicyc 1 e max), was determined ergometer as described previously (4).
Body composition: Percentage bodyfat measured with an underwater weighing culated (4)
was derived from body density which was technique. Lean body mass was then cal-
Blood sampling and preparation of plasma: Blood collection was performed in the morning hours (to exclude diurnal variations) during the follicular phase of the menstrual cycle and between days 1-14 of the hormone-induced cycle. All blood samples were drawn in upright position from the antecubital vein by clean venipuncture, using a 1.2 mm bore needle. The first 2 ml were discarded and the next 9 ml were collected in a plastic tube containing 1 ml of 3.8% trisodium citrate-2-hydrate for the assay of the haemostatic factors. Citrated blood was immediately centrifuged (4 x 103g) for 10 min at room temperature. Platelet poor plasma was divided into small aliquots, snap frozen and stored at -7O'C to be assayed.
Assay of coagulation factors: Plasma antigen levels of FII, PC, total PS, PSfree and C4b-bp were determined by Laurel1 rocket immunoelectrophoresis. Gels for the Laurel1 plates contained 0.9% agarose (Seakem, Rockland ME, USA), 8 mmol/l EDTA(Sigma,St Louis MO, USA) and 2% PEG 8000 (Sigma, St Louis MO, USA) in 0.08 mol/l Tris/0.025 mol/l Tricine buffer pH 8.6. The same buffer with 8 mmol/l EDTA was employed for electrophoresis. To the gels 0.75% anti-F11 (Clotimmun-Prothrombin, Behringwerke, Marburg, FRG), 0.7% anti-PS (see below) and 1% anti-C4b-bp (Behring Diagnostics, La Jolla Ca, USA) were added for the assays of FII, PS and C4b-bp respectively. Staining of the plates was performed with Coomassie Brilliant Blue R 250 (Merck, Darmstadt, FRG). PC antigen levels were measured using a commercially available test kit (Assera.plate protein C, Diagnostica Stago, Asnieres, France), according to the instructions of the manufacturer. Each Laurel1 plate was standardized with four dilutions (25, 50, 75, 100%) of a normal plasma pool (n=20). Two dilutions of each plasma sample were analyzed and the antigen levels were calculated and expressed relative to the normal pool which was defined as 100%. For the separation of the complexed form of PS (PS-C4b-bp) and the free form of PS (PS-free) a technique was used based on the method recently described by Comp et al (18). Addition of PEG 8000 to plasma in a final concentration of 4.2% resulted in the precipitation of PS-Cbb-bp, while PS-free remained in the supernatant as was confirmed by crossed immunoelectrophoresis (18). Laurel1 plates containing 0.7% anti-PS were used to analize the supernatant. A standard
Vol. 45, No. 1
ORAL CONTRACEPTIVES & PROTEIN S
111
curve from normal pooled plasma that had been treated in the same way was included in each plate. Electrophoresis for total PS (free and bound PS) and PS-free was conducted at 25 'C with 5V/cm for 15-18 hrs. Under these conditions a single peak for total PS was observed. Anti-PS was prepared in rabbits using purified PS (19). The antiserum gave one precipitation line when tested in double immunodiffusion against normal plasma. This line showed a reaction of identity with the precipitation line obtained against purified PS. Anti-PS inhibited the cofactor activity of PS in normal plasma (20).
Statistical analysis: Means and standard deviations were calculated with the usual methods. Date were compared by Student's t-test or by Mann-Whitney test as appropriate. Differences were considered statistically significant at P
RESULTS
The effect of OCA on PC and PS was studied in two groups of highly trained athletes. The.groups were very well matched, no significant differences between users and controls were observed in age (23.7 + 1.8 and 23.4 5 2.2 year), weight (69.3 + 4.8 and 70.8 + 6.3 kg), body fat (24.7 + 4.3 and 23.3 2 2.5%) and physical conditioning (3.84 2 0.44 and 4.18 + 0.32 irO;-max l/min). In the group of OCA-users a significant lower PS antigen level (P
TABLE I Antigen levels of Plasma Proteins
Controls (n=lO> Factor II Protein C Protein S C4-b-binding protein Protein S-free
112.7 + 108.4 T 111.7 T 96.4 + 104.6 T
17.2 20.4 21.8 13.8 15.2
Users (n=lO) 113.2 + 109.7 T 84.5 + 81.5 T 94.3 z
13.6 33.0 19.0 * 14.0 * 18.0
Values expressed as % of normal pooled plasma. Means + SD are presented. * Significant differences between the groups P
In plasma PS circulates partly in a complex with C4b-bp. The level of C4b-bp therefore, affects the level of PS-free. In the group of users a significantly decreased level of C4b-bp (P
112
ORAL CONTRACEPTIVES & PROTEIN S
Vol.
45, No. 1
DISCUSSION
Despite a gradual reduction in oestrogens and progestagens, the use of OCA is still associated with an enhanced risk for thromboembolism in a number of women (1). It has been established that the observed venous thromboembolic complications were strongly related to the oestrogen dose of OCA (2). A comparable association has been reported between the oestrogen content and the magnitude of the changes observed in the levels of vitamin K-dependent clotting factors (3, 4, 5). The present investigation was undertaken following the recent publication of studies reporting a clear relation between thromboembolic disease and depressed levels of either PC (15) or PS (16). The effect of low-dose OCA on FII, PC, PS and C4b-bp was studied in healthy equally well-conditioned women. The most striking observations in our study were the reduced levels of PS and C4b-bp in relation to OCA medication. In view of the association between low PS plasma levels and venous thrombosis, our findings might well explain, at first sight, the enhanced incidence of thromboembolic complications reported in users of OCA. The interpretation of changes in (total) PS is, however, complicated by the fact that only a fraction of the protein (PS-free) is active as a cofactor for the anticoagulant effect of activated PC (14). The major part of PS bound to C4b-bp, an inhibitory protein of the complement system, is functionally inactive (18). C4b-bp has a potential regulatory function with respect to the activity of PS, since changes in the plasma levels of this protein determine the distribution between bound and free forms of PS. In our study the OCArelated reduction in total PS was paralleled by a reduction in C4b-bp. The compensatory changes in C4b-bp resulted in normal PS-free plasma levels that were fully comparable to those observed in the controls. It is quite conceivable that subjects who fail to maintain the optimal equilibrium between PS and Clrb-bp plasma levels might be prone to an enhanced thrombogenecity. Interesting in this respect are the reported shifts from free to bound PS in pregnancy, nephrotic syndrome and lupus erythematosus, conditions that have been associated with thromboembolic complications (21).The close relation between the complement and the coagulation system is stressed by earlier studies (4,22) that reported OCA-induced reductions in Cl-inactivator, another plasma protein involved in both systems. OCA-related increases in the plasma levels of FII and PC, as reported previously (3, 5), were not observed in the group of users. The fact that the subjects in our study were well-conditioned and used OCA with a low oestrogen content might explain the discrepancy. From the results of our study we conclude that a reduction in the plasma level of total PS is not necessarily associated with a lower level of free, functionin the group of users ally active PS. The normal levels of PC and PS-free suggest that OCA medication has no adverse effect on the protein C system in healthy individuals. The OCA-related enhanced fibrinolytic potential observed in a comparable group of athletes (4) could support this conclusion.
REFERENCES
1. BuTTIGER,L.E., thromboembolic 101,198O.
BOMAN,G., EKLUND,G. and WESTERHOLM,B. Oral contraceptives and disease: effect of lowering estrogen content. Lancet 1,1097-
2. MEADE,T.W. Oral contraceptives, Gynaecols,758-61,1982.
clotting factors and thrombosis.Am
J Obstet
Vol. 45, No. 1
ORAL CONTRACEPTIVES & PROTEIN S
113
3.
MAMMEN,E.F. Oral contraceptives and blood coagulation:a critical review. Am J Obstet GynecolE,781-790,1982.
4.
HUISVELD,I.A., KLUFT,C., HOSPERS,J.E.H., BERNINK,M.J.E., ERICH,W.B.M. and BOUMA,B.N. Effect of exercise and oral contraceptive agents on fibrinolytic potential in trained females. J Appl Physiol REEP x,906-13,1984.
5.
MEADE,T.W., STIRLING,Y.,WILKES,H. and MANNUCC1,P.M. Effects of oral contraceptives and obesity on protein C antigen. Thromb Haemost =,198-9,1985.
6.
STENFLO,J. A new vitamin K-dependent protein: purification from bovine plasma and preliminary characterization. J Biol Chem =,355-63,1976.
7.
DI SCIPIO,R.G., HERMODSON,M.A., YATES,S.G. and DAVIE,E.W. A comparison of human prothrombin, factor IX, factor X and protein S. Biochemistry *,698706,1977.
8.
MARLAR,R.A., KLEISS,A.J. and GRIFFIN,J.H. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 2,1067-72,1982.
9.
CLOUSE,L.H. and COMP,P.C. The regulation of haemostasis: the protein C system. N Engl J Med x,1298-304,1986.
10.
COMP,P.C. and ESMON,C.T. Generation of fibrinolytic activity by infusion of activated protein C into dogs. J Clin Invest E,1221-8,198l.
11.
TAYLOR,F.B. and LOCKHART,M.S. Whole blood clot lysis: in vitro modulation by activated protein C. Thromb Res x,639-49,1984.
12.
DE FOUW,N.J., HAVERKATE,F., BERTINA,R.M., KOOPMAN,J.,VAN WIJNGAARDEN,A. and VAN HINSBERGH,V.W.M. The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro. -Blood 67,118992,1986.
13.
DAHLB#CK,B. and STENFLO,J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Nat1 Acad Sci USAz,2512-6,198l.
14.
WALKER,F.J. Regulation of activated protein C by a new protein: a possible function for bovine protein S. J Biol Chem 255, 5521-4,198O.
15.
GRIFFIN,J.H., EVATTS,B., ZIMMERMAN,T.S., KLEISS,A.J. and WIDEMAN,C. Deficiency of protein C in congenital thrombotic disease.J Clin Invest 68,1370-3,1982.
16.
SCHWARZ,H.P., FISCHER,M., HOPMEIER,P., BATARD,M.A. and GRIFFIN,J.H. Plasma protein S deficiency in familial thrombotic disease. -Blood 311,1525-8,1984.
17.
WALLEWIS,J.L., BARTON,T., STEAD,N.W., WILLIAMS,R.S., LOGUE,E.E., LACE,A.G.and PIZZ0,S.V. Physical conditioning augments the fibrinolytic response to venous occlusion in healthy adults. N Engl J Med =,98791,198O.
18.
COMP,P.C., DORAY,D., PATTON,D.and ESMON,C.T. An abnormal plasma distribution of protein S occurs in functional protein S deficiency.Blood 67,504-8,1986.
114
ORAL CONTRACEPTIVES & PROTEIN S
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