1 Integrative Vascular Biology Laboratory, Department of Kinesiology and Applied Physiology, University of Colorado, Boulder, Colorado 80309; and Divisions of 2 Cardiology and 3 Geriatrics, Department of Medicine, University of Colorado, Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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We determined the influence of oral
contraceptives (OC) on the capacity of the endothelium to release
tissue-type plasminogen activator (t-PA). Twenty-three healthy
premenopausal women were studied: 12 nonusers and 11 users of OC. Net
endothelial release rates of t-PA were calculated as the product of the
arteriovenous concentration gradient and forearm plasma flow in
response to intra-arterial bradykinin (BK: 12.5-50
ng · 100 ml
tissue1 · min
1) and
sodium nitroprusside (SNP: 1.0-4.0
µg · 100 ml
tissue
1 · min
1). Net
release of t-PA antigen and increment in t-PA activity across the
forearm to BK increased (P < 0.01) in a dose-dependent fashion and to similar extents in the nonusers and users of OC. At the
highest BK dose, net release of t-PA antigen was 64.5 ± 8.2 and
66.2 ± 15.4 ng · 100 ml
tissue
1 · min
1 in the
nonusers and users of OC, whereas the net increment in t-PA activity
was 18.6 ± 3.0 and 16.0 ± 2.0 IU · 100 ml
tissue
1 · min
1,
respectively. There was no effect of SNP on t-PA release in either
group. These results indicate that endothelial t-PA release is not
altered in premenopausal women who use oral contraception.
fibrinolysis; endothelium; tissue-type plasminogen activator; bradykinin; contraceptives
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INTRODUCTION |
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IT IS ESTIMATED THAT MORE than 100 million women worldwide use oral contraception (42). Clinical and epidemiological data indicate that oral contraceptive use is associated with an increased risk of thrombotic disorders, including myocardial infarction, deep vein thrombosis, and venous thromboembolism (16, 39, 40, 41). In fact, the risk of thrombosis has been reported to be two- to fourfold greater in users compared with nonusers of oral contraceptives, independent of smoking status (15, 41). The mechanisms underlying this apparent prothrombotic state are not clear. Considerable attention has focused on the coagulation-fibrinolysis axis, specifically, whether the procoagulant effects of oral contraceptives are balanced by increased fibrinolytic capacity.
Several studies have reported that oral contraceptives lower
circulating plasma concentrations of tissue-type plasminogen activator
(t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen and
activity, and increase levels of t-PA activity, suggesting that
fibrinolytic activity is increased with oral contraceptive use
(14, 20, 29). However, favorable alterations in
circulating levels of t-PA and PAI-1 induced by oral contraceptive use
may not necessarily reflect increased fibrinolytic activity per se but
rather changes in hepatic clearance (20). Moreover, recent data indicate that it is the capacity of the endothelium to release t-PA rapidly and acutely from intracellular storage pools and not
circulating plasma fibrinolytic concentrations that determines the
efficacy of endogenous fibrinolysis (13, 35, 40a). Indeed, thrombolysis is more effective if active t-PA is readily
available during, rather than after, thrombus formation (1,
9). Furthermore, the inhibitory interaction between PAI-1 and
t-PA has a second-order rate constant of
~107 · M1 · s
1
(33); therefore, local rapid release of t-PA is critical
to the fibrinolytic process. Although the effects of oral
contraceptives on plasma markers of fibrinolysis have been well
studied, little information is available regarding the impact of oral
contraceptives on endothelial t-PA release.
Accordingly, the aim of the present investigation was to determine the influence of oral contraceptive use on the capacity of the vascular endothelium to release t-PA in healthy premenopausal women. To address this aim, we employed an isolated forearm model to determine, in vivo, rates of endothelial t-PA release in well-matched healthy premenopausal women who were either taking or not taking oral contraceptives.
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MATERIALS AND METHODS |
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Subjects.
Twenty-three healthy sedentary premenopausal women were studied: 12 nonusers (age 21-40 yr) and 11 users of oral contraceptives (22-38 yr). All women were nonobese, normotensive (blood pressure <140/90), nonsmokers, eumenorrheic, and free of overt disease, as
assessed by medical history and fasting blood chemistries. Women not
using oral contraceptives had discontinued use for at least 1 yr before
the start of the study. The women taking oral contraceptives had done
so continuously for at least 6 mo (range: 0.5-12 yr) before the
study (Table 1). Five of the 11 women
using oral contraception were taking second-generation and six were taking third-generation oral contraceptives. All women were studied during the follicular phase of their menstrual cycle. In addition, all
subjects were free of recent infection/inflammation (<2 wk) as
determined by questionnaire (27). Before participation,
all of the subjects had the research study and its potential risks and
benefits explained fully before providing written informed consent
according to the guidelines of the University of Colorado at Boulder.
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Body composition. Body mass was measured to the nearest 0.1 kg using a medical beam balance (Detecto, Webb City, MO). Percent body fat was determined by dual-energy X-ray absorptiometry (model DPX-IQ; Lunar Radiation, Madison, WI). Body mass index was calculated as weight (kilograms) divided by height (meters) squared.
Metabolic measurements. Fasting plasma lipid and lipoprotein and glucose and insulin concentrations were determined using conventional methods by the clinical laboratory affiliated with the General Clinical Research Center, as previously described (6).
Treadmill exercise test. To assess aerobic fitness, subjects performed incremental treadmill exercise using a modified Balke protocol as previously described by our laboratory (6). Maximal oxygen consumption was measured using on-line computer-assisted open-circuit spirometry. In addition, heart rate and rating of perceived exertion were measured throughout exercise, and total exercise time to exhaustion was recorded.
Experimental protocol.
All measurements were performed between 7:00 and 10:00 AM after a 12-h
overnight fast in a temperature-controlled room. An intravenous
catheter was placed in a deep antecubital vein of the nondominant arm.
Thereafter, a 5-cm 20-gauge catheter was introduced in the brachial
artery of the same arm under local anesthesia (1% lidocaine). After
catheterization, the subjects were allowed to rest 20 min before
baseline measurements were made. Resting heart rate and intra-arterial
pressure were recorded during this period and were monitored
continuously throughout the infusion protocol. Forearm blood flow (FBF)
was measured using strain-gauge venous occlusion plethysmography
(D. E. Hokanson, Bellevue, WA), as previously described by our
laboratory (7). Drug infusion rates were normalized per
100 ml forearm tissue and infused at 4 ml/min by a syringe pump. After
the measurement of resting blood flow for 5 min, bradykinin was infused
intra-arterially at 12.5, 25, and 50 ng · 100 ml
tissue1 · min
1 and
sodium nitroprusside at 1.0, 2.0, and 4.0 µg · 100 ml
tissue
1 · min
1 for 5 min at each dose. To avoid an order effect, the sequence of drug
administration was randomized.
Blood sampling and biochemical assays.
All phlebotomies were performed with minimal venostasis. Arterial and
venous blood samples were collected simultaneously at baseline and the
end of each drug dose to determine t-PA and PAI-1 antigen and activity
concentrations. The first 3 ml of blood from both the artery and vein
were discarded before each sample was collected. Blood samples were
collected in tubes containing 0.45 M sodium citrate buffer, pH 4.3 (final dilution volume 1:10; Stabilyte; Biopool). Within 15 min of
collection, all samples were centrifuged for 20 min at 6,000 g at 4°C. Platelet-poor plasma was separated into aliquots
and stored at 70°C until assayed at the end of the study. All
assays were performed in duplicate with a maximum of one freeze-thaw
cycle. Plasma concentrations of t-PA and PAI-1 antigen and activity as
well as factor VII-antigen (Ag) were determined by enzyme immunoassay
(Biopool International, Ventura, CA, and Diagnostic, Stago, France).
t-PA activity is expressed in international units, and PAI-1 activity
is expressed in arbitrary units.
Net release of fibrinolytic factors across the forearm.
Net endothelial release of t-PA and PAI-1 antigen and increment in t-PA
and PAI-1 activity in response to bradykinin and sodium nitroprusside
was calculated according to Jern et al. (13). Briefly,
arteriovenous concentration gradients were determined by subtracting
the measured values in simultaneously collected venous and arterial
blood. For both t-PA and PAI-1, a positive difference indicated a net
release, and a negative difference indicated net uptake. Net release or
uptake rates were calculated as follows: net release = (CV CA) × [FBF × (101
hematocrit/100)], where
CV and CA represent the
concentration in the vein and artery, respectively. Hematocrit was
measured in triplicate using the standard microhematocrit technique and
corrected for trapped plasma volume within the trapped erythrocytes
(3). The total amount of t-PA antigen released and total
increment in t-PA activity across the forearm in response to bradykinin
were calculated as the total area under each curve above baseline using
a trapezoidal model.
Statistical analysis. Differences in subject baseline characteristics and area under the curve were determined by ANOVA. Differences between groups in net release of fibrinolytic factors across the forearm in response to incremental intra-arterial doses of bradykinin and sodium nitroprusside were determined by repeated-measures ANOVA. There were no significant differences in any of the key outcome variables between users of second- and third-generation oral contraceptives. Therefore, the data were pooled and presented together. All data are reported as means ± SE. Statistical significance was set a priori at P < 0.05 for all comparisons.
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RESULTS |
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Subject characteristics.
Table 1 presents selected subject characteristics. There were no
significant differences in anthropometric, hemodynamic, or metabolic
factors between the groups. Baseline venous plasma concentrations of
fibrinolytic and coagulation factors are shown in Table
2. Circulating concentrations of t-PA
antigen, PAI-1 antigen, and PAI-1 activity were lower (all
P < 0.05) in the users compared with nonusers of oral
contraceptives. There was no difference in t-PA activity between the
groups. Additionally, plasma levels of factor VII-Ag were significantly
higher in the users than nonusers of oral contraception.
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Vasodilator and fibrinolytic responses to bradykinin and sodium
nitroprusside.
As shown in Fig. 1, the FBF responses to
bradykinin and sodium nitroprusside were almost identical between the
groups. There were no significant group differences in basal
endothelial net release of t-PA antigen (1.6 ± 1.1 vs. 0.4 ± 0.9 ng · 100 ml
tissue1 · min
1,
P = 0.44) or increment in t-PA activity (0.4 ± 0.2 vs. 0.4 ± 0.1 IU · 100 ml
tissue
1 · min
1,
P = 0.71) between the women taking and not taking oral
contraceptives. In response to bradykinin, the net release of t-PA
antigen and increment in t-PA activity increased in a dose-dependent
fashion and to similar extents in the users of oral contraceptives
compared with nonusers. At the highest bradykinin dose (50 ng · 100 ml tissue
1 · min
1), net
endothelial release of t-PA antigen was 66.2 ± 15.4 ng · 100 ml
tissue
1 · min
1 in the
users compared with 64.5 ± 8.2 ng · 100 ml
tissue
1 · min
1 in the
nonusers (Fig. 2), whereas the peak
increment in t-PA activity was 16.4 ± 1.7 and 18.6 ± 3.0 IU · 100 ml
tissue
1 · min
1,
respectively (Fig. 3). In addition, there
were no group differences in either the total amount of t-PA antigen
released (area under the curve: 299 ± 72 vs. 327 ± 40 ng/100 ml tissue; P = 0.73) or the total increment in
t-PA activity (88 ± 10 vs. 101 ± 14 IU/100 ml tissue;
P = 0.46) across the forearm in response to bradykinin. The infusion of sodium nitroprusside caused no changes in endothelial t-PA release in either group (Figs. 2 and 3). The effects of bradykinin and sodium nitroprusside on PAI-1 release (antigen and activity) were
minimal and not significantly different between the groups. For
example, at the highest dose of bradykinin, net release of PAI-1
antigen was 2.3 ± 1.3 ng · 100 ml
tissue
1 · min
1 in the
users compared with 2.4 ± 2.2 ng · 100 ml
tissue
1 · min
1 in the
nonusers of oral contraceptives.
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DISCUSSION |
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The primary new finding of the present investigation is that endothelial regulation of fibrinolysis appears to be unaffected by oral contraceptive use. We observed similar rates of basal and stimulated endothelial release of t-PA antigen and increment in t-PA activity across the forearm in healthy premenopausal women taking and not taking oral contraceptives. In addition, although endothelial t-PA release was not different between the groups, the women taking oral contraceptives demonstrated higher circulating levels of coagulation factor VII-Ag. It has been suggested that the procoagulant effects of oral contraceptives are counterbalanced by increased fibrinolytic potential (5, 28). The results of the present study question this postulate.
Premenopausal women who take oral contraceptives are at an increased risk of arterial and venous thrombosis (38-41). Although the precise mechanisms for the enhanced thrombotic risk associated with oral contraceptive use have not been completely elucidated, inadequate fibrinolysis as a causal factor has generally been dismissed. In fact, a number of studies have shown that oral contraceptive use is associated with favorable changes in plasma fibrinolytic markers suggestive of enhanced fibrinolytic activity (14, 20, 29). Our finding of lower plasma concentrations of t-PA antigen, PAI-1 antigen, and PAI-1 activity in the users compared with nonusers of oral contraceptives is in line with these previous reports. However, changes in steady-state plasma fibrinolytic concentrations provide an indirect and, in some cases, misleading assessment of endothelial t-PA release and, in turn, endogenous fibrinolytic potential (12, 13). Oral contraceptive-induced reductions in plasma t-PA and PAI-1 concentrations may reflect estrogen-associated changes in hepatic synthesis and clearance and not enhanced fibrinolysis (20). For example, estrogen has been reported to increase t-PA clearance through upregulation of the manose receptor, a hepatic clearance receptor for t-PA (23). In addition, the ineffectiveness of transdermal compared with oral estrogen administration in lowering basal PAI-1 levels in postmenopausal women supports the concept that the hepatic effects of estrogen are important in the regulation of PAI-1 synthesis and clearance (10, 22).
The salient and novel finding of the present study is that the capacity of the endothelium to acutely release t-PA is unaffected by oral contraceptive use. The magnitude of increase in net endothelial release of t-PA antigen and increment in t-PA activity across the forearm in response to bradykinin was not different between the women taking and not taking oral contraceptives and, importantly, was similar to levels previously reported in healthy young adults (2). Moreover, we observed no significant changes in t-PA release in response to sodium nitroprusside, indicating that the increases observed with bradykinin were not a blood flow-related phenomenon. Our in vivo findings are consistent with in vitro studies demonstrating no effect of estradiol, ethinyl estradiol, levonorgestrel, or gestodene on endothelial t-PA production (21). Considering t-PA is the key enzyme in initiating an endogenous fibrinolytic response, absence of greater endothelial t-PA release in premenopausal women taking oral contraceptives argues against the notion of increased fibrinolytic activation with oral contraceptive use.
Increased coagulation is thought to be a primary factor in the increased thrombotic risk associated with oral contraceptives. Plasma concentrations of several markers of coagulation, including factor VII, VIII, and X and fibrinogen, have been shown to be higher and activity of the protein C pathway lower in users compared with nonusers of oral contraceptives (20, 26, 30). Our finding of significantly higher levels of factor VII-Ag in users compared with nonusers of oral contraceptives is in accordance with these previous reports. Importantly, however, despite evidence of increased coagulability, we observed no compensatory increase in endothelial t-PA release associated with oral contraceptive use in the present study. This finding is in line with that of Meijers et al. (29) who reported no effect of oral contraceptives on overall clot-lysis time, despite favorable changes in plasma fibrinolytic factors suggesting enhanced fibrinolytic activity. Collectively, the results of the present study and Maijers et al. (29) suggest that the procoagulant effects of oral contraceptives are not neutralized by enhanced fibrinolytic capacity. An imbalance between coagulation and fibrinolysis may indeed underlie the increased thrombotic risk associated with oral contraceptives.
It is noteworthy that none of the premenopausal women in the present study were smokers. Cigarette smoking has been shown to profoundly impair endothelial t-PA release throughout the vasculature (31, 32, 36). For example, Newby and colleagues (31, 32) reported that current and ex-smokers release significantly less t-PA in the forearm and coronary circulation compared with nonsmokers. In addition, reduced coronary t-PA release was associated with increased atheromatous plaque burden (31). This is particularly relevant given the fact that premenopausal women who smoke and take oral contraceptives are at 10-30 times greater risk of thrombotic events compared with their nonsmoking counterparts (37, 41, 43). It is likely that if smokers were included in the present study our results would have been different.
In addition to fibrinolytic function, the vascular endothelium plays an important role in the regulation of vascular tone through the synthesis and release of various vasodilating and vasoconstricting substances (24). One of the most important vasodilating substances released by the endothelium is nitric oxide (NO). Impaired NO-mediated endothelium-dependent vasodilation is thought to contribute to the etiology of atherosclerosis and thrombosis (4, 25). In the present study, the FBF responses to the endothelial agonist bradykinin were not significantly different between the users and nonusers of oral contraceptives. Our results compliment those of John et al. (17) who reported no impairment in ACh-mediated endothelium-dependent vasodilation in a similar group of healthy premenopausal women taking oral contraceptives. Both bradykinin and ACh are NO-dependent endothelial agonists; however, they stimulate endothelium-dependent vasodilation via different receptor-mediated mechanisms (34). Thus, taken together, these findings suggest that the vascular risk associated with oral contraceptive use does not appear to involve impaired endothelial vasodilator function.
Three experimental limitations of the present study should be mentioned. First, with all cross-sectional studies, it is possible that genetic and/or other lifestyle behaviors influenced the results of our group comparisons. We attempted to minimize potential lifestyle influences by studying healthy women across the premenopausal age range who were nonsmokers and did not differ in body composition or habitual physical activity. Second, potential regional differences in fibrinolytic activity limit extrapolation of our findings in the forearm to other vascular beds (11, 18). However, there is evidence to suggest that forearm endothelial t-PA release may be an excellent surrogate measure of t-PA release in the coronary vasculature (31, 32). Nevertheless, the results of the present study should be viewed within the context of the vascular bed studied. Finally, differences in thrombotic risk have been reported between oral contraceptives containing second-generation progestagens (primarily levonorgestrel) and third-generation progestagens (gestodene and desogestrel; see Refs. 38 and 39). A recent meta-analysis reported a 1.7-fold increased risk of venous thrombosis in women using third-generation compared with second-generation oral contraceptives (19). The mechanisms responsible for this increased risk are not clear. Although we observed no difference in t-PA release between the users of second- and third-generation oral contraceptives (total t-PA antigen released: 256 ± 76 vs. 333 ± 122 ng/min, P = 0.62), in the present study the small sample size, associated variability, and lack of statistical power limit interpretation. Future studies are needed to determine the independent effects of second- and third-generation oral contraceptives on endothelial t-PA release.
In conclusion, the results of the present study indicate that oral contraceptive use does not influence, either positively or negatively, endothelial t-PA release in healthy premenopausal women. The lack of a compensatory increase in endogenous endothelial fibrinolytic capacity to counteract enhanced coaguability may contribute mechanistically to the increased thrombotic risk associated with oral contraceptive use.
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ACKNOWLEDGEMENTS |
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We thank all of the women who participated in the study and Marilyn Ng and Sharon Blackett for technical assistance.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-03840, an American Diabetes Association Clinical Research Award, and American Heart Association Awards 0060430Z (C. A. DeSouza), 0120679Z (B. L. Stauffer), and 0110220Z (D. T. Smith).
Address for reprint requests and other correspondence: C. DeSouza, Integrative Vascular Biology Laboratory, Dept. of Kinesiology and Applied Physiology, Univ. of Colorado, 354 UCB, Boulder, CO 80309 (E-mail: desouzac{at}colorado.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 20, 2002;10.1152/ajpendo.00333.2002
Received 23 July 2002; accepted in final form 18 August 2002.
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