Physical Activity Status and Adverse Age-Related Differences in Coagulation and Fibrinolytic Factors in Women

From the Human Cardiovascular Research Laboratory, Center for Physical Activity, Disease Prevention, and Aging, Department of Kinesiology, University of Colorado, Boulder (C.A.D., P.P.I., D.R.S.); and the Department of Medicine, Divisions of Cardiology and Geriatric Medicine, University of Colorado, Health Sciences Center, Denver (D.R.S.).

Correspondence to Christopher DeSouza, PhD, Department of Kinesiology, University of Colorado, Campus Box 354, Boulder, CO 80309. E-mail desouzac{at}stripe.Colorado.edu

	Abstract

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Abstract—Adverse changes in coagulation and fibrinolytic factors are thought to contribute to the increased risk of cardiovascular disease and atherothrombosis with age. We tested the hypothesis that such age-related changes in specific coagulation and fibrinolytic factors are absent in physically active women. Resting levels of plasma fibrinogen, tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) antigen and activity, and fibrin D-dimer were measured in 24 healthy premenopausal women: 11 sedentary (aged 28±1 years; Pre-S) and 13 physically active (aged 30±1 years; Pre-PA) and in 27 healthy postmenopausal women: 14 sedentary (aged 61±1 years; Post-S) and 13 physically active (aged 58±1 years; Post-PA). Post-S had higher (P<.05) fibrinogen, t-PA antigen, PAI-1 antigen, PAI-1 activity, and fibrin D-dimer levels and lower t-PA activity than Pre-S. Post-PA demonstrated lower (P<.01) plasma fibrinogen, t-PA antigen, PAI-1 antigen, and PAI-1 activity and higher (P<.01) t-PA activity levels than Post-S. In addition, plasma fibrin D-dimer levels tended (P=.06) to be lower in Post-PA than in Post-S. Although plasma levels of fibrinogen and fibrin D-dimer in Post-PA were lower than in Post-S, they were higher (P<.05) than in Pre-PA. Importantly, however, the fibrinolytic profile of Post-PA did not differ from that of Pre-PA. The results of the present study demonstrate that the adverse age-associated differences in plasma fibrinogen concentrations and the endogenous fibrinolytic system in sedentary healthy women are either attenuated or absent in highly physically active women. The smaller or absent age-related differences in coagulation and fibrinolytic factors in women who habitually exercise may represent an important mechanism contributing to their lower age-related increase in both cardiovascular disease and atherothrombotic events. Future studies need to determine whether women who are moderately active would demonstrate the same favorable hemostatic profile.

Key Words: aging • exercise • fibrinogen • fibrinolytic system • fibrin D-dimer

	Introduction

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Advancing age is associated with an increased risk of CVD in general and atherosclerotic vascular disease in particular.¹ In women, the incidence of both CVD and thrombosis increases after the onset of menopause.^{2 3} It has been suggested that age-related changes in coagulation and fibrinolytic factors contribute to the increased risk of atherothrombotic events in postmenopausal women by accelerating the atherosclerotic process and promoting thrombus formation.^{4 5 6} Indeed, higher plasma concentrations of fibrinogen⁴ and fibrin D-dimer,⁷ both markers of thrombogenic risk, and reduced endogenous fibrinolytic activity⁵ have been reported in healthy postmenopausal compared with premenopausal women.

In contrast to aging, regular physical activity is associated with favorable coagulation and fibrinolytic function.^{8 9 10} We have previously shown that physically active postmenopausal women demonstrate lower plasma fibrinogen⁸ concentrations and improved fibrinolytic function evidenced by lower t-PA antigen and PAI-1 activity⁹ levels compared with age-matched sedentary control subjects. However, whether regular physical activity prevents adverse age-related changes in coagulation and fibrinolytic factors is unknown. If so, it could contribute to the smaller age-related increases in both CVD and atherothrombotic events observed in physically active compared with sedentary women.¹¹

Accordingly, the primary purpose of the present investigation was to test the hypothesis that adverse age-related differences in specific coagulation and fibrinolytic factors are absent in physically active women. To systematically test this hypothesis, we used a cross-sectional model to first document the detrimental age-associated differences in plasma fibrinogen, t-PA antigen, t-PA activity, PAI-1 antigen, PAI-1 activity, and fibrin D-dimer levels in healthy sedentary women. We then measured the aforementioned hemostatic factors in corresponding groups of endurance-trained women.

	Methods

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Subjects
Fifty-one healthy women were studied: 11 Pre-S, 13 Pre-PA, 14 Post-S, and 13 Post-PA women. The Pre-PA and Post-PA women were matched for age-adjusted running performance as described previously by our laboratory¹² and ran 58±7 and 48±3 km/wk, respectively. The physically active women were recruited from various running clubs throughout the Boulder area and from participants in the Bolder Boulder, the second largest 10-km road race in the United States. The sedentary subjects were recruited through local newspaper advertisements and had not participated in a regular aerobic exercise program for at least 1 year before the start of the study. All of the postmenopausal women were at least 2 years postmenopausal (range, 2 to 23 years), and an equal number of each group were taking estrogen-based hormone supplements (8 sedentary and 8 physically active). All premenopausal women were eumenorrheic, as assessed by self-report of menstrual cycles, not taking oral contraceptives, and studied during the follicular phase of their menstrual cycle. All subjects were free of overt disease, as assessed by medical history. The postmenopausal women were further evaluated for clinical evidence of cardiovascular disease with a physical examination and resting and maximal exercise electrocardiograms. No subjects were on medication other than hormone replacement, and all subjects were nonsmokers. In addition, the subjects did not differ in alcohol consumption, as assessed by 4-day dietary records. 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.

Body Composition
Body mass was measured to the nearest 0.1 kg by using a medical beam balance (Detecto). Total body density was determined by hydrodensitometry, with residual volume measured by oxygen dilution.¹³ Body fat percentage was calculated using the equation of Brozek et al.¹⁴ Fat-free mass (kg) was calculated from percent body fat and body mass. The WHR was calculated as the ratio of the minimal waist circumference to the circumference of the maximal gluteal protuberance. Waist circumference was measured at the narrowest part of the torso and was used as an estimate of abdominal adiposity.¹⁵

Maximal Oxygen Consumption (VO₂max)
VO₂max was determined by using an on-line computer-assisted open-circuit spirometry during incremental exercise on a motorized treadmill as previously described.¹² Expired O₂ and CO₂ gas fractions were measured by using a Perkin-Elmer MGA-1100 mass spectrometer, and expired volume was determined by using a turbine (VMM-2, Interface Associated). A valid VO₂max was accepted when at least three of the following criteria were met: (1) a plateau in VO₂ with increasing work rate (<1 mL/kg^-1/min or <100 mL/min), (2) a respiratory exchange ratio at maximal exercise >1.10, (3) achievement of age-predicted maximal heart rate (220 minus age), and (4) a rating of perceived exertion >18 (Borg Scale).¹⁶

Blood Sampling and Preparation
To avoid the diurnal variation in coagulation and fibrinolytic variables, all blood samples were collected between 7:30 AM and 10:30 AM after a 12-hour overnight fast. All phlebotomies were performed with minimal venostasis. The first 2 to 3 mL of blood was discarded, and samples were used only if venous return was prompt throughout. Blood for determination of fibrinogen, t-PA antigen, t-PA activity, and fibrin D-dimer was collected in syringes containing 1.0 mL of 130 mmol/L sodium citrate (final dilution volume 1:10). To prevent in vitro inactivation of t-PA by ongoing complex formation with PAI-1, 0.75 mL of citrate-anticoagulated whole blood was acidified within 1 minute of phlebotomy by addition of 0.37 mL of 0.5 mmol/L sodium acetate, pH 4.2. Blood samples to measure PAI-1 antigen and PAI-1 activity were collected in syringes containing modified Files solution (1 mL acid-citrate-dextrose solution, 80 µL acetylsalicylic acid solution, and 10 µL PGE1 solution)¹⁷ to minimize in vitro platelet activation (final dilution volume 1:5). Within 30 minutes of phlebotomy, all samples were centrifuged for 20 minutes at 6000g at 4°C. Platelet-poor plasma was aliquoted and stored at -60°C until assayed at the end of the study. All assays were performed in duplicate with a maximum of one freeze-thaw cycle. Intra-assay variability was calculated from duplicate samples, and internal controls were used to determine interassay variability.

A questionnaire designed to detect and document recent infection/inflammation (<2 weeks) was administered before the phlebotomies. Subjects with a history of recent infection/inflammation did not receive phlebotomy to avoid confounding effects from potential infection/inflammation-associated hemostatic changes.¹⁸

Measurement of Coagulation and Fibrinolytic Variables
Plasma fibrinogen levels were measured using the method of Clauss.¹⁹ Plasma t-PA antigen, PAI-1 antigen, and fibrin D-dimer were determined by using an enzyme-linked immunosorbent assay (American Bioproducts). t-PA activity and PAI-1 activity were measured using an amidolytic method (Chromogenix). t-PA activity is expressed in international units and PAI-1 activity in arbitrary units. One AU is defined as the amount of inhibitor that inhibits 1 IU of t-PA per milliliter of plasma.²⁰ Intra-assay and interassay coefficients of variation were 8.6% and 7.5%, respectively, for t-PA antigen; 6.9% and 7.4% for t-PA activity; 8.9% and 8.2% for PAI-1 antigen; 3.8% and 3.5% for PAI-1 activity; and 4.7% and 6.4% for fibrin D-dimer.

Statistical Analysis
The influence of both age and physical activity on all variables was determined by a multifactor ANOVA (agexphysical activity). When indicated by a significant F value, specific mean comparisons were performed to identify significant group differences. Simple and forward stepwise multiple regression analyses and partial correlation coefficients were calculated to determine relations between the specific coagulation and fibrinolytic variables and anthropometric, hemodynamic, and metabolic variables. ANCOVA was used to statistically adjust for the influence of each independent determinant identified by the stepwise multiple regression analysis. All data are expressed as mean±SE. Statistical significance was set at P<.05.

	Results

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Physical Characteristics
Table 1 shows the physical characteristics of the subjects. Body mass, percent body fat, and BMI were higher (P<.05) in Pre-S and Post-S than in their age-matched physically active counterparts. In addition, percent body fat and BMI were higher (P<.01) in Post-S than in Pre-S and Post-PA than in Pre-PA. There were no differences in fat-free mass among the four groups. WHR was higher (P<.01) in Post-S than in any other group. The Pre-PA demonstrated the highest (P<.01) and Post-S the lowest (P<.01) VO₂max of all groups. Systolic blood pressure was higher (P<.01) in the postmenopausal women relative to their respective premenopausal control subjects. There were no differences in diastolic blood pressure among the four groups.

Coagulation and Fibrinolytic Variables
Fibrinogen
There was a significant main effect of both age and physical activity on plasma fibrinogen levels. The age-related difference was apparent in both the sedentary and physically active women, as plasma fibrinogen levels were higher in Post-S than in Pre-S (2.85±0.09 versus 2.28±0.08 g/L; P<.01) and Post-PA than in Pre-PA (2.49±0.09 versus 2.18±0.07 g/L; P=.02; Fig 1). The physical activity–related difference, however, was observed only in the older subjects, with lower (P=.002) plasma fibrinogen levels in Post-PA than in Post-S.

View larger version (14K): [in this window] [in a new window]   Figure 1. Plasma fibrinogen (A) and fibrin D-dimer (B) concentrations in the Pre-S, Post-S, Pre-A, and Post-PA groups. Values are mean±SE. *P<.05 vs premenopausal women of same physical activity status. P<.01 vs age-matched sedentary.

Fibrinolytic System
Fig 2 shows the significant age- and physical activity–related differences in the fibrinolytic system. Post-S had higher (P<.05) t-PA antigen (8.0±0.9 versus 4.0±0.5 ng/mL), PAI-1 antigen (9.8±1.8 versus 4.8±1.4 ng/mL), and PAI-1 activity (14.8±1.2 versus 11.5±1.2 AU/mL) and lower t-PA activity (1.3±0.1 versus 2.2±0.3 IU/mL) levels than Pre-S. In addition, the molar concentration ratio of active t-PA to active PAI-1 also was higher (P<.05) in Post-S (1:13.8±2.2 mmol/L) than in Pre-S (1:7.3±1.8 mmol/L). In contrast, Post-PA appeared to have avoided the adverse age-associated differences in the fibrinolytic system, as t-PA antigen (3.9±0.6 ng/mL), PAI-1 antigen (1.9±0.4 ng/mL), and PAI-1 activity (6.5±1.1 AU/mL) were all lower (P<.01) and t-PA activity (2.7±0.4 IU/mL) higher (P<.01) in Post-PA than in Post-S but were not different in Post-PA versus Pre-PA. Consequently, the molar concentration ratio of active t-PA to active PAI-1 also was lower (P<.01) in Post-PA (1:3.0±0.7 mmol/L) relative to Post-S and almost identical to that of Pre-PA (1:2.2±0.5 mmol/L). Although Pre-PA demonstrated the lowest absolute levels of t-PA antigen (2.8±0.4 ng/mL), PAI-1 antigen (1.2±0.1 ng/mL), and PAI-1 activity (5.0±1.2 AU/mL) and the highest levels of t-PA activity (2.9±0.3 IU/mL) of all the groups, only PAI-1 antigen, PAI-1 activity, and the molar concentration ratio of active t-PA to active PAI-1 differed significantly compared with the Pre-S group.

View larger version (20K): [in this window] [in a new window]   Figure 2. Plasma t-PA antigen (A), t-PA activity (B), PAI-1 antigen (C), and PAI-1 activity (D) levels in the Pre-S, Post-S, Pre-PA, and Post-PA groups. Values are mean±SE. *P<.01 vs premenopausal women of same physical activity status. P<.01 vs age-matched sedentary.

Fibrin D-Dimer
Similar to fibrinogen, plasma fibrin D-dimer levels were higher in Post-S than in Pre-S (335.0±35.6 versus 113.0±11.1 ng/mL) and Post-PA than in Pre-PA (265.2±24.6 versus 123.7±11.4 ng/mL; Fig 1). However, although the Post-PA tended (P=.06) to have lower plasma fibrin D-dimer levels compared with the Post-S, there was no significant effect of physical activity status on plasma fibrin D-dimer levels.

Correlations and ANCOVA
Significant univariate correlations were observed between the specific coagulation and fibrinolytic variables and body composition, blood pressure, and metabolic characteristics of the subjects (Table 2). Multiple stepwise regression analysis revealed that percent body fat (R²=.31) and plasma total cholesterol (R²=.31) were the primary correlates of plasma fibrinogen and fibrin D-dimer concentrations in the overall study population, respectively. For the fibrinolytic variables, percent body fat (R²=.51) and BMI (R²=.23) were the strongest correlates of t-PA antigen and t-PA activity, respectively whereas BMI (R²=.53) and waist circumference (R²=.46) were the strongest correlates of PAI-1 antigen and PAI-1 activity, respectively. After statistically controlling for each primary determinant, the differences among the groups were no longer significant.

	Discussion

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There are three important findings of the present study. First, although physical activity status does not appear to prevent an age-related increase in plasma fibrinogen concentrations, the magnitude of the elevation with age is only 50% as great in habitually exercising compared with sedentary women. Second, adverse age-related differences in the fibrinolytic system were not observed in the physically active women, suggesting that regular physical activity may prevent the decline in fibrinolytic function observed with age in sedentary women. Finally, similar age-related increases in plasma fibrin D-dimer levels were observed in both the physically active and sedentary women.

Fibrinogen
Plasma fibrinogen, the circulating precursor of fibrin, is a major independent risk factor for atherosclerotic CVD in postmenopausal women.^{4 5} Plasma fibrinogen levels have been shown to increase with age^{21 22} and to be related to the incidence of future coronary events in both healthy and diseased populations.⁶ In addition, we⁸ and others¹⁰ have previously reported that plasma fibrinogen concentrations are lower in physically active than in sedentary postmenopausal women. The present findings confirm and extend these earlier observations by demonstrating that the age-related elevation in plasma fibrinogen levels is twice as great in the sedentary (+0.57 g/L) as in the physically active (+0.31 g/L) women. Importantly, the lower plasma fibrinogen levels in the physically active women would appear to be associated with a reduction in CVD risk. Epidemiological data from the Framingham study indicated that among women, each SD (0.55 g/L) increment in plasma fibrinogen levels increased the risk of an initial cardiovascular event by 25%.²³ Thus, although there was a significant increase in plasma fibrinogen levels with age in both the sedentary and physically active groups in the present study, the magnitude of the increase and the associated cardiovascular risk appears to be lower in physical activity women. Lower plasma fibrinogen concentrations may reduce the risk of atherosclerosis and thrombosis by favorably altering blood viscosity and platelet adhesion and aggregation^{24 25} and by limiting intravascular fibrin formation and deposition.^{26 27}

The mechanisms by which habitual physical activity may attenuate the age-related increase in plasma fibrinogen concentrations are not clear. It has been suggested that the favorable association between plasma fibrinogen levels and regular exercise are likely due, at least in part, to lower body fatness.^{28 29} Indeed, in the present study, percent body fat was the primary determinant of plasma fibrinogen level, accounting for approximately one third of the variability.

Fibrinolytic System
The hemostatic mechanism responsible for the proteolytic degradation of intravascular fibrin deposition is the fibrinolytic system. This enzymatic pathway maintains vascular patency by converting the inactive proenzyme plasminogen to the active enzyme plasmin, which lyses fibrin into soluble degradation products.³⁰ Both clinical and epidemiological data have suggested that reduced endogenous fibrinolytic activity, characterized by increased t-PA antigen, PAI-1 antigen, and PAI-1 activity and reduced t-PA activity, is a major contributor to both the development and severity of atherothrombosis.^{31 32 33 34}

Our finding of impaired fibrinolytic function with age in sedentary women is consistent with previous reports²² and supports the hypothesis that hypofibrinolysis may contribute to the increased risk of atherothrombotic events with age in women.³³ In addition, the lower t-PA antigen and PAI-1 activity observed in the physically active postmenopausal women relative to the sedentary control subjects confirm our previous observations in middle-aged and older women.³⁵ Importantly, the results of the present study significantly extend our previous findings by showing, for the first time, that the adverse age-related differences in the fibrinolytic system observed in sedentary women are absent in physically active women. In contrast to their sedentary peers, there were no age-related increases in t-PA antigen, PAI-1 antigen, and PAI-1 activity or a concomitant decrease in t-PA activity in the physically active women. In fact, the hyperfibrinolytic state observed in the physically active postmenopausal women was similar to that observed in their active premenopausal peers. As a result, the molar concentration ratio of active t-PA to active PAI-1, an index of fibrinolytic potential defined as the ability to respond to a stimulus and lyse fibrin,³⁶ was almost identical in the premenopausal and postmenopausal endurance-trained women.

Thus, these data suggest that adverse age-related differences in the fibrinolytic system may not be a primary effect of aging. Rather, such changes may be due, at least in part, to age-related reductions in physical activity and associated increases in body weight and fatness. In the present study, percent body fat, BMI, and waist circumference were the primary determinants of the fibrinolytic variables. These observations are consistent with previous studies that have reported an association between the fibrinolytic system and total and abdominal body fatness^{37 38} and support the suggested link between adiposity and thrombogenic risk.^{38 39}

In the present study, the lower t-PA antigen, PAI-1 antigen, and PAI-1 activity and the higher t-PA activity observed in the physically active compared with sedentary postmenopausal women may be of significant clinical importance. Recent findings suggest that t-PA antigen is both a biological marker of subclinical atherosclerosis⁴⁰ and a strong predictor of future acute myocardial infarction³¹ in healthy individuals. In addition, because t-PA is synthesized primarily by endothelial cells, it has been proposed that elevated levels may also reflect endothelial cell inflammation and damage.⁴⁰ Furthermore, PAI-1, the major determinant of fibrinolytic activity,³⁰ has been suggested to play an important role in the development of atherothrombosis.³² Elevated PAI-1 gene expression, localization, and production have been reported to occur in injured atherosclerotic arteries.⁴¹ In contrast, elevated t-PA activity is associated with a decreased risk of cardiovascular events.⁴² Thus, the absence of age-associated impairments in the fibrinolytic system in the physically active women may be mechanistically involved in the lower incidence of atherothrombotic events observed in this population.¹¹

Fibrin D-Dimer
Fibrin D-dimer is a specific degradation product of the enzymatic action of plasmin on cross-linked fibrin.⁴³ Elevated plasma levels have been suggested to be a sensitive marker of intravascular fibrin formation⁴³ and the extent and severity of underlying atherosclerosis.^{44 45} In the present study, there was a significant influence of age on plasma fibrin D-dimer concentrations in both the sedentary and physically active women. Our finding of higher plasma fibrin D-dimer levels in the sedentary postmenopausal compared with premenopausal women is consistent with previous studies^{7 22} and supports the premise of increased fibrin formation and deposition with age.^{45 46} An original finding of the present study, however, was that regular physical activity does not appear to influence the effect of age on plasma fibrin D-dimer levels. Although the physically active postmenopausal women demonstrated lower absolute plasma fibrin D-dimer levels than their sedentary age-matched peers, they had significantly higher fibrin D-dimer levels than their younger counterparts. However, given the fact that the age-related increase in plasma fibrin D-dimer levels in the postmenopausal groups occurred under two very different fibrinolytic conditions, ie, low fibrinolytic activity in the sedentary group and high fibrinolytic activity in the physically active group, it is possible that the elevated levels of fibrin D-dimer may be reflecting two different physiological conditions. In the sedentary postmenopausal women, the age-related increase in plasma fibrin D-dimer levels may reflect a prothrombotic state characterized by numerous small amounts of fibrin degradation products resulting from chronic extensive intravascular fibrin formation and degradation.³⁴ On the other hand, the elevated levels of plasma fibrin D-dimer found in our physically active postmenopausal group may reflect increased fibrin turnover resulting from elevated levels of circulating fibrin.⁴⁴

Limitations
The primary limitation of this study is its cross-sectional design and the inherent possibility that constitutional factors may have influenced our findings. However, the plasma concentrations of the hemostatic factors presented herein are consistent with previous studies involving both sedentary^{7 47 48} and physically active¹⁰ populations. In addition, the favorable plasma levels of fibrinogen, t-PA antigen, t-PA activity, and PAI-1 activity associated with regular physical activity in the present study are similar to those reported in response to endurance exercise training in older men.⁴⁹ Nevertheless, to determine the effects of physical activity per se on coagulation and fibrinolytic factors in postmenopausal women, longitudinal studies in this population will need to be performed. Second, it is important to note that the physically active subjects in the present study were highly endurance-trained athletes; it was our purpose to use these women to address the physiological question posed. As such, we are unable to comment on whether women who are moderately active would demonstrate the same favorable hemostatic profile. Given the clinical importance of coagulation and fibrinolysis to thrombogenic risk, future studies are needed to address this issue.

Conclusion
In conclusion, the results of the present study indicate that the adverse age-associated differences in plasma fibrinogen concentrations and the endogenous fibrinolytic system in sedentary healthy women are either attenuated or absent in highly physically active women. These differences may play an important role in the smaller age-related increase in the incidence of CVD and thrombosis in physically active compared with sedentary women.

	Selected Abbreviations and Acronyms

 

AU = arbitrary units BMI = body mass index CVD = cardiovascular disease PAI-1 = plasminogen activator inhibitor-1 Pre-PA = premenopausal physically active Pre-S = premenopausal sedentary Post-PA = postmenopausal physically active Post-S = postmenopausal sedentary t-PA = tissue-type plasminogen activator WHR = waist-to-hip ratio

	Acknowledgments

 
This research was supported by NIH RO1 awards HL-39966, AG-06537, and AG-13038 (D.R. Seals); Research Supplement to Minority Individuals in Postdoctoral Training awards RO1 HL-39966 (C.A. DeSouza) and AG-13038 (C.A. DeSouza); and NIH F32 award AG-05705 (P.P. Jones). We would like to thank all of the subjects who participated in the study, the General Clinical Research Center at the University of Colorado Health Science Center, Mary Jo Reiling for her technical assistance, and Cyndi Long for her administrative assistance.

Received April 22, 1997; accepted October 30, 1997.

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