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Hemostatic response to acute physical exercise in healthy adolescents
Journal of Science and Medicine in Sport (2007) 10, 164—169
ORIGINAL PAPER
Hemostatic response to acute physical exercise in healthy adolescents J. Ribeiro a, A. Almeida-Dias b, A. Ascens˜ ao c, J. Magalh˜ aes c, A.R. Oliveira a, J. Carlson d,∗, J. Mota c, H.-J. Appell e, J. Duarte b,c a
EsEF/UFRGS Physical Education School, Federal University of Rio Grande do Sul, Porto Alegre, Brazil North Politecnic Institute of Health, CESPU, CRL, Gandra, Paredes, Portugal c CIAFEL, Faculty of Sports Sciences, University of Porto, Portugal d Centre for Ageing, Rehabilitation, Exercise & Sport, Victoria University, PO Box 14428, Melbourne 8001, Australia e Department of Physiology and Anatomy, German Sport University Cologne, Germany b
Received 31 May 2006; accepted 1 June 2006 KEYWORDS Endothelium; Coagulation; Fibrinolysis; Adolescent exercise
∗
Summary The chronic and immediate post-exercise responses in the hemostatic and fibrinolytic systems have been shown to be variable and reflect differing adaptations with ageing and responses to exercise protocols. This study investigated the effects of acute and exhaustive exercise on the amplitude and duration of hemostatic and fibrinolytic responses in young adolescent males. The sample comprised 10 sedentary boys (13.2 ± 0.5 years, 55.8 ± 11.3 kg, 165.7 ± 7.4 cm), who had not exercised or received any medication for at least 2 weeks before the experiments. The subjects performed exhaustive stepping exercise, consisting of 1 s up and down cycles to fatigue. When the subjects were unable to maintain the required stepping rhythm, they were given a 30 s recovery period. Following each 30 s recovery participants recommenced the stepping cadence until fatigue prevented them continuing. Venous blood samples were drawn before and immediately, 1 and 24 h after exercise to assess the following coagulation and fibrinolytic parameters: Platelet counts, activated partial thromboplastin time (aPTT), prothrombin time (PT), coagulation factor VIII (FVIII:C), von Willebrand factor (vWF), fibrinogen concentration, thrombin—antithrombin complex (TAT), D-dimer, plasminogen activator inhibitor (PAI-1), and tissue-type plasminogen activator (t-PA). Immediately following exercise, platelet counts, aPTT, FVIII, vWF and t-PA were significantly elevated in contrast to PAI-1, which decreased significantly until 1 h after exercise. FVIII and platelet counts were elevated at 1 and 24 h after exercise, respectively. Only the parameters FVIII and PAI-1 did not return to baseline values during the first hour after physical exercise. When compared to adults the results revealed different rates and ranges of coagulation and fibrinolysis parameters being activated by exhaustive exercise in this group of adolescents. © 2006 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +61 3 9919 4111; fax: +61 3 9919 4539. E-mail address: [email protected] (J. Carlson).
1440-2440/$ — see front matter © 2006 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsams.2006.06.001
Hemostatic response to acute physical exercise in healthy adolescents
Introduction The tendency for a post-exercise hypercoagulation state has been documented revealing higher circulating platelet counts, shorter activated partial thromboplastin time (aPTT) and a higher activity of factor VIII (FVIII).1,2 Concomitantly, there appears to exist a global increase in fibrinolytic activity, expressed by an increase in tissue plasminogen activator (t-PA) and a decrease in plasminogen activator inhibitor (PAI-1) circulating levels.3—6 However, many factors such as experimental design, exercise protocol, training status, health status, and methods of analysis, seem to interfere with the expression of markers for these two systems. Response data reported in the literature are often conflicting7,8 especially concerning the magnitude of the response and the time necessary (varying from 1 to 24 h after exercise) to recover from acute exercise-induced alterations.2,6,9 Most studies examining the coagulation and fibrinolytic systems have been conducted on adults, in which arterial degenerative diseases could be well established as a consequence of their ageing.10 Endothelial dysfunction is a key factor in this atherosclerotic process, and markers of endothelial abnormalities have been sought, particularly those involved in a disturbed endothelium-dependent vasomotion or in a liberation of cell molecular products.11 Since von Willebrand factor (vWF), t-PA and PAI-1 play an important role in hemostasis and are released by endothelial cells, they are commonly used as markers for endothelial dysfunction.11 Epidemiological evidence indicates that elevated plasma concentrations of these markers are potent predictors of both myocardial infarction and stroke incidence.12 Since aging is associated with adverse changes in coagulation and fibrinolysis, suggesting a thrombophilic state,13 it is important to distinguish the modifications of the hemostatic system induced by acute exercise and those induced by ageing related endothelial impairment. In fact, as referred above, advanced age may influence the behaviour of many hemostatic parameters, determining responses such as, an increase in platelet aggregatability, an increase in PAI-1 plasma levels, and a decrease in t-PA activity, the latter probably associated with degenerative vascular diseases.14 Thus, because the hemostatic responses to acute physical exercise in adults may be influenced by age-dependent endothelial dysfunction,10 this study proposed to investigate these responses in young people not yet showing endothelial dysfunction, but already equipped with a mature hemostatic system. Limited research has examined the
165
influence of acute exercise on the hemostatic system in young populations who already possess cardiovascular risk factors such as obesity.15 Therefore the aim of this study was to investigate the effects of acute and exhaustive exercise on the amplitude and duration of the hemostatic and fibrinolytic responses in young adolescent males.
Material and methods Subjects and experimental protocol Following the receipt of informed consent from their parents, 10 healthy sedentary boys (age = 13.2 ± 0.5 years, weight = 55.8 ± 11.3 kg, height = 165.7 ± 7.4 cm) volunteered to participate in this study. The subjects were required to refrain from having performed any type of heavy physical exercise or having received any medication for at least 2 weeks prior to the commencement of the study. The subjects were required to perform an exhaustive exercise consisting of stepping up and down from a step. The step height was individually adjusted to the height of each subject’s femoral condyles. The stepping rhythm was paced acoustically at 60 beats per minute with same periods (1 s) for stepping up and down, respectively. When a subject was not able to maintain the required stepping rhythm, he was given a 30 s recovery period. Following the 30 s recovery he was required to recommence another set of the stepping cadence until fatigued and subsequently given another 30 s recovery period. This sequence was continued until complete exhaustion when even after a 30 s recovery the subject was not able to continue. The total number of sets and the stepping repetitions in each set were recorded for each subject (Table 1).
Sampling and hemostatic parameters Blood samples were collected before, immediately at fatigue, 1 and 24 h after the exercise. Blood (10 ml) was withdrawn by puncture from the antecubital vein and immediately collected in polypropylene tubes with anticoagulant and selected parameters measured using the following techniques. Platelet counts were performed with an electronic particle counter (Thrombocyte Analyser 147C-Analys Instrument) on whole blood kept in EDTA (1 mg/ml). Partial thromboplastin time (aPTT) and prothrombine time (PT) estimations were performed by citrate plasma analysis KC4 instrument (Amelunk, Germany) using appropriate reagents
166
J. Ribeiro et al.
Table 1
Mean value of repetitions number (Rep) performed in each one of the 21 sets of exercise protocol
Sets 1 Rep 172 N 10
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 21
58 10
73 10
52 10
47 10
41 10
41 10
31 9
31 8
26 8
35 7
27 6
20 6
18 5
18 4
18 4
16 4
12 3
13 1
7 1
4 1
The numbers of subjects (n) that executed each set are also presented.
Table 2 Means and standard deviations of hemostatic parameters observed at different moments of the experimental protocol Parameter studied
Time after exercise Before
Platelets (x109/l) vWF (U/l) aPTT (s) PT (s) TAT (g/l) Fibrinogen (g/l) FVIII (U/l) t-PA (g/l) PAI-1 (g/l) D-Dimer ((g/l) * **
276 0.89 32.7 12.3 1.9 2.8 1.16 3.6 28.2 95.9
± ± ± ± ± ± ± ± ± ±
0h 50 0.18 4.7 0.8 0.8 0.8 0.4 1.4 12.1 62.4
318 0.97 27.8 12.4 2.2 3.0 1.58 8.4 23.8 234.4
1h ± ± ± ± ± ± ± ± ± ±
50** 0.17** 4.3* 1.0 1.4 0.8 0.64* 5.1* 8.0* 262.2
283 0.89 30.0 12.6 3.4 2.8 1.33 3.3 19.6 137.8
24 h ± ± ± ± ± ± ± ± ± ±
45 0.17 4.1 1.0 4.7 0.8 0.36* 1.7 8.6* 116.5
303 0.87 33.0 12.5 2.5 2.8 1.04 3.7 26.5 153.5
± ± ± ± ± ± ± ± ± ±
46** 0.12 4.8 1.0 2.0 0.7 0.44 3.4 12.0 195.6
p < 0.05 vs. before. p < 0.01 vs. before.
(Diagnostic Grifolds SA). FVIII:C coagulant activity was assayed by the synthetic chromogenic substrate method using COATEST® Factor VIII (Chromogenix AB, Molndal, Sweden). von Willebrand factor (vWF) was measured using an enzyme-linked immunosorbent assay (ELISA), IMUBIND® vWF (American Diagnostica Inc., USA). Fibrinogen concentration was determined by the Clauss method with the respective reagents (Diagnostic Grifolds SA). For measuring thrombin-antithrombin complex (TAT) we developed an ELISA method using both capture antibody (TAT-EIA-C) and detecting antibody (TAT-EIA-D) from Affinity Biologicals Inc. (Enzyme Research Laboratories Inc., USA). D-dimer was evaluated by a solid phase enzyme immunoassay COALIZA® D-Dimer (Chromogenix AB, Molndal, Sweden). Plasminogen activator inhibitor (PAI-1) and tissue plasminogen activator (t-PA) were determined by IMUBIND® total t-PA and IMUBIND® Plasma PAI-1 ELISA kit (American Diagnostica Inc., USA), respectively.
Results
Statistical analysis
Discussion
Absolute values and percentages of variations based on pre-exercise values were expressed as means with standard deviations. Differences of means were tested with ANOVA for repeated measures, and the level of significance was set at ˛ < 0.05.
Exercise-dependent effects on the coagulant activity of the FVIII/vWF complex have been studied with various exercise protocols of varying intensities and durations. Despite these differences in experimental protocols, studies with healthy adults sug-
The mean number of exercise sets performed was 14.0 ± 5.0, and the total time of exercise was 1289 ± 294 s. The total number of sets, the mean number of repetitions in each set, and the number of subjects that performed every set are shown in Table 1. The absolute values for the studied hemostatic parameters are presented in Table 2, whilst the percentages of variation from the pre-exercise values are depicted in Fig. 1. Immediately after exercise, platelet counts, aPTT, FVIII, vWF and t-PA were significantly increased, while in contrast PAI-1 was significantly lower. At 1 and 24 h after the exercise, only FVIII and platelet counts, respectively, remained significantly elevated. At 1 h after exercise the PAI-1 was still significantly reduced.
Hemostatic response to acute physical exercise in healthy adolescents
167
Figure 1 Exercise effect on platelets, von Willebrand factor (vWF), activated partial thromboplastin time (aPTT), coagulation factor VIII (FVIII), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Values are expressed as means of percentage of variation from the values obtained before exercise (=100%).
gest significant increases in coagulant activity.2,3,14 Compared to adult experiments, the present study, with adolescent males also showed significant increase in FVIII/vWF concentrations immediately after exercise. In adult studies conducted at varying intensities, results have shown that FVIII activity can increase considerably, with concomitant variations of the vWF.5,9 In the present study however, despite the exhaustive exercise, the adolescent males demonstrated relatively lower enhancements in coagulant activity with FVIII increasing 36% and vWF increasing by 8.9%. These results concur with other studies that demonstrate FVIII increases are of greater magnitude when compared with those of vWF.5,6,9 Following exhaustive exercise in adults that consisted of dynamic or isometric contractions, the hypercoagulation response is also supported by the observed shortening of aPTT (7—38%).16 The PT response to exercise remains controversial, with several studies demonstrating both, significant shortening,9 and non-significant differences.1,3 The effect of acute exercise on plasma fibrinogen concentrations in adults is equivocal. Several studies demonstrated no differences,3,4 while others revealed significant increases17 or even significant decreases6 in plasma fibrinogen. The healthy adolescents of the present study produced a 15% shortening of aPTT immediately after exercise, while PT, TAT, and fibrinogen did not change. In contrast with adult responses in which the aPTT and PT changes seem to be persistent until 1 and 24 h after exercise, respectively,18 the present study revealed that aPTT returned to baseline after 1 h, and PT did not show any alteration.
The enhancement of FVIII and vWF, and the concomitant shortening of aPTT observed in this study suggest an activation of the coagulant system following exhaustive exercise. However, this activation seems to be less pronounced than observed in adult studies with exhaustive exercise.5,9 In addition, the response time of coagulant activity of adolescents appears to differ to that observed in adults. A plausible explanation for these responses can be related to differences in the ‘‘maturity status’’ of the responsible coagulant mechanisms, such as endothelial functionality. As the stimulus responsible for exercise-induced increases in plasma vWF and FVIII content seems to be mediated by -adrenergic receptors through a nitric oxide-dependent mechanism, the hemostastic system could be conditioned by endothelial function and be modified during the aging process.19 At rest, the aging-dependent endothelial dysfunction is usually associated with a basal thrombophilic state, i.e. an increased coagulation tendency with concomitantly decreased fibrinolytic activities.13 However, with physical exercise, an enhancement of fibrinolytic activity was observed, with increased plasmin formation resulting from the endothelial release of t-PA with a parallel decrease in PAI-1.3,4 Increases in fibrinolytic activities ranging from 75 to 250%, when compared to baseline levels, appear influenced by exercise intensity.5 In this study, the t-PA increased by 133% and PAI-1 decreased by 15.6% immediately after acute exercise and are consistent responses associated with a major activation of the fibrinolytic system in these healthy adolescents. Following short maximal exercise, increases in D-dimer con-
168 centrations (further marker of hyperfibrinolysis) have been measured in adults,1 but in this group of adolescents there was no significant increase in this parameter. The time to full post-exercise recovery to return to the resting levels of fibrinolytic parameters is equivocal, since several studies have registered 45—60 min after intense exercise,9 2 h after long distance running,2 or even 24 h after a marathon race.6 In our study, the t-PA returned to the resting levels 1 h after exercise while PAI-1 showed a significant decrease (30%) at 1 h post and was only normalized after 24 h. Previous studies in healthy adults and older subjects demonstrated that a single bout of exercise produced a pronounced increase in endogenous fibrinolysis, which may be protective against exertionrelated atherothrombotic events.20 However, an enhanced tendency for thrombosis may offset the increased fibrinolytic effect of exercise reported in adults.11 In this study, healthy adolescents showed a modest increase of coagulant activity and a more pronounced increase of fibrinolytic activity that might represent a powerful protection against thrombogenic events. Compared to adults, this difference in the thrombogenic response observed in adolescent males could be explained by the differing endothelial functionality associated with ageing. Whilst the results of the current study reveal desirable hemostatic responses observed in healthy adolescents, a logical extension of the current study would seem to question whether examining ‘‘well trained’’ adolescents might reflect the potential cumulative effects that both youth and fitness might have on beneficial hemostatic function.
Practical implications • This study provides normal adolescent values of hemostatic responses of adolescent males. • Developmentally, adolescent boys demonstrate no detrimental changes in their hemostatic system. • When compared to adults, the results reveal adolescents show different rates and ranges of coagulation and fibrinolysis parameters being activated by exhaustive exercise.
Acknowledgement The first author (JR) was kindly supported by CAPES, Brazil.
J. Ribeiro et al.
References 1. Molz AB, Heyduck B, Lill H, et al. The effect of different exercise intensities on the fibrinolytic system. Eur J Appl Physiol 1993;67:298—304. 2. Hansen JB, Wilsgard L, Olsen JO, et al. Formation and persistence of procoagulant and fibrinolytic activities in circulation after strenuous physical exercise. Thromb Haemost 1990;64:385—9. 3. El-Sayed MS, Lin X, Rattu AJ. Blood coagulation and fibrinolysis at rest and in response to maximal exercise before and after a physical conditioning programme. Blood Coagul Fibrinol 1995;6:747—52. 4. Rankinen T, Vaisanen S, Penttila I, et al. Acute dynamic exercise increases fibrinolytic activity. Thromb Haemost 1995;73:281—6. 5. Andrew M, Carter C, O’Brodovich H, et al. Increases in factor VIII complex and fibrinolytic activity are dependent on exercise intensity. J Appl Physiol 1986;60: 1917—22. 6. Prisco D, Paniccia R, Bandinelli B, et al. Evaluation of clotting and fibrinolytic activation after protracted physical exercise. Thromb Res 1998;89:73—8. 7. Womack CJ, Nagelkirk PR, Coughlin AM. Exercise-induced changes in coagulation and fibrinolysis in healthy populations and patients with cardiovascular disease. Sports Med 2003;33:795—807. 8. El-Sayed MS, El-Sayed Ali Z, Ahmadizad S. Exercise and training effects on blood haemostasis in health and disease: an update. Sports Med 2004;34:181—200. 9. Ferguson EW, Bernier LL, Banta GR, et al. Effects of exercise and conditioning on clotting and fibrinolytic activity in men. J Appl Physiol 1987;62:1416—21. 10. Matz RL, Andriantsitohaina R. Age-related endothelial dysfunction: potential implications for pharmacotherapy. Drugs Aging 2003;20:527—50. 11. Raitakari OT, Celermajer DS. Testing for endothelial dysfunction. Ann Med 2000;32:293—304. 12. Gallistl S, Sudi KM, Borkenstein M, et al. Determinants of haemostatic risk factors for coronary heart disease in obese children and adolescents. Int J Obes Relat Metab Disord 2000;24:1459—64. 13. van den Burg PJ, Hospers JE, van Vliet M, et al. Changes in haemostatic factors and activation products after exercise in healthy subjects with different ages. Thromb Haemost 1995;74:1457—64. 14. van den Burg PJ, Hospers JE, Mosterd WL, et al. Aging, physical conditioning, and exercise-induced changes in hemostatic factors and reaction products. J Appl Physiol 2000;88:1558—64. 15. Gallistl S, Sudi KM, Cvirn G, et al. Effects of shortterm energy restriction and physical training on haemostatic risk factors for coronary heart disease in obese children and adolescents. Int J Obes Relat Metab Disord 2001;25:529—32. 16. Hyers TM, Martin BJ, Pratt DS, et al. Enhanced thrombin and plasmin activity with exercise in man. J Appl Physiol 1980;48:821—5. 17. Torres-Guerra E, Diez-Ewald M, Vizcaino G, et al. Effect of stress electrocardiogram on platelet function, concentration of von Willebrand factor and fibrinogen in hypertensive patients and healthy subjects. Invest Clin 2000;41: 105—16. 18. Rocker L, Taenzer M, Drygas WK, et al. Effect of prolonged physical exercise on the fibrinolytic system. Eur J Appl Physiol 1990;60:478—81.
Hemostatic response to acute physical exercise in healthy adolescents 19. Jilma B, Dirnberger E, Eichler HG, et al. Partial blockade of nitric oxide synthase blunts the exercise-induced increase of von Willebrand factor antigen and of factor VIII in man. Thromb Haemost 1997;78:1268—71.
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20. Dufaux B, Order U, Liesen H. Effect of a short maximal physical exercise on coagulation, fibrinolysis, and complement system. Int J Sports Med 1991;12:S38—42.
ORIGINAL PAPER
Hemostatic response to acute physical exercise in healthy adolescents J. Ribeiro a, A. Almeida-Dias b, A. Ascens˜ ao c, J. Magalh˜ aes c, A.R. Oliveira a, J. Carlson d,∗, J. Mota c, H.-J. Appell e, J. Duarte b,c a
EsEF/UFRGS Physical Education School, Federal University of Rio Grande do Sul, Porto Alegre, Brazil North Politecnic Institute of Health, CESPU, CRL, Gandra, Paredes, Portugal c CIAFEL, Faculty of Sports Sciences, University of Porto, Portugal d Centre for Ageing, Rehabilitation, Exercise & Sport, Victoria University, PO Box 14428, Melbourne 8001, Australia e Department of Physiology and Anatomy, German Sport University Cologne, Germany b
Received 31 May 2006; accepted 1 June 2006 KEYWORDS Endothelium; Coagulation; Fibrinolysis; Adolescent exercise
∗
Summary The chronic and immediate post-exercise responses in the hemostatic and fibrinolytic systems have been shown to be variable and reflect differing adaptations with ageing and responses to exercise protocols. This study investigated the effects of acute and exhaustive exercise on the amplitude and duration of hemostatic and fibrinolytic responses in young adolescent males. The sample comprised 10 sedentary boys (13.2 ± 0.5 years, 55.8 ± 11.3 kg, 165.7 ± 7.4 cm), who had not exercised or received any medication for at least 2 weeks before the experiments. The subjects performed exhaustive stepping exercise, consisting of 1 s up and down cycles to fatigue. When the subjects were unable to maintain the required stepping rhythm, they were given a 30 s recovery period. Following each 30 s recovery participants recommenced the stepping cadence until fatigue prevented them continuing. Venous blood samples were drawn before and immediately, 1 and 24 h after exercise to assess the following coagulation and fibrinolytic parameters: Platelet counts, activated partial thromboplastin time (aPTT), prothrombin time (PT), coagulation factor VIII (FVIII:C), von Willebrand factor (vWF), fibrinogen concentration, thrombin—antithrombin complex (TAT), D-dimer, plasminogen activator inhibitor (PAI-1), and tissue-type plasminogen activator (t-PA). Immediately following exercise, platelet counts, aPTT, FVIII, vWF and t-PA were significantly elevated in contrast to PAI-1, which decreased significantly until 1 h after exercise. FVIII and platelet counts were elevated at 1 and 24 h after exercise, respectively. Only the parameters FVIII and PAI-1 did not return to baseline values during the first hour after physical exercise. When compared to adults the results revealed different rates and ranges of coagulation and fibrinolysis parameters being activated by exhaustive exercise in this group of adolescents. © 2006 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +61 3 9919 4111; fax: +61 3 9919 4539. E-mail address: [email protected] (J. Carlson).
1440-2440/$ — see front matter © 2006 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsams.2006.06.001
Hemostatic response to acute physical exercise in healthy adolescents
Introduction The tendency for a post-exercise hypercoagulation state has been documented revealing higher circulating platelet counts, shorter activated partial thromboplastin time (aPTT) and a higher activity of factor VIII (FVIII).1,2 Concomitantly, there appears to exist a global increase in fibrinolytic activity, expressed by an increase in tissue plasminogen activator (t-PA) and a decrease in plasminogen activator inhibitor (PAI-1) circulating levels.3—6 However, many factors such as experimental design, exercise protocol, training status, health status, and methods of analysis, seem to interfere with the expression of markers for these two systems. Response data reported in the literature are often conflicting7,8 especially concerning the magnitude of the response and the time necessary (varying from 1 to 24 h after exercise) to recover from acute exercise-induced alterations.2,6,9 Most studies examining the coagulation and fibrinolytic systems have been conducted on adults, in which arterial degenerative diseases could be well established as a consequence of their ageing.10 Endothelial dysfunction is a key factor in this atherosclerotic process, and markers of endothelial abnormalities have been sought, particularly those involved in a disturbed endothelium-dependent vasomotion or in a liberation of cell molecular products.11 Since von Willebrand factor (vWF), t-PA and PAI-1 play an important role in hemostasis and are released by endothelial cells, they are commonly used as markers for endothelial dysfunction.11 Epidemiological evidence indicates that elevated plasma concentrations of these markers are potent predictors of both myocardial infarction and stroke incidence.12 Since aging is associated with adverse changes in coagulation and fibrinolysis, suggesting a thrombophilic state,13 it is important to distinguish the modifications of the hemostatic system induced by acute exercise and those induced by ageing related endothelial impairment. In fact, as referred above, advanced age may influence the behaviour of many hemostatic parameters, determining responses such as, an increase in platelet aggregatability, an increase in PAI-1 plasma levels, and a decrease in t-PA activity, the latter probably associated with degenerative vascular diseases.14 Thus, because the hemostatic responses to acute physical exercise in adults may be influenced by age-dependent endothelial dysfunction,10 this study proposed to investigate these responses in young people not yet showing endothelial dysfunction, but already equipped with a mature hemostatic system. Limited research has examined the
165
influence of acute exercise on the hemostatic system in young populations who already possess cardiovascular risk factors such as obesity.15 Therefore the aim of this study was to investigate the effects of acute and exhaustive exercise on the amplitude and duration of the hemostatic and fibrinolytic responses in young adolescent males.
Material and methods Subjects and experimental protocol Following the receipt of informed consent from their parents, 10 healthy sedentary boys (age = 13.2 ± 0.5 years, weight = 55.8 ± 11.3 kg, height = 165.7 ± 7.4 cm) volunteered to participate in this study. The subjects were required to refrain from having performed any type of heavy physical exercise or having received any medication for at least 2 weeks prior to the commencement of the study. The subjects were required to perform an exhaustive exercise consisting of stepping up and down from a step. The step height was individually adjusted to the height of each subject’s femoral condyles. The stepping rhythm was paced acoustically at 60 beats per minute with same periods (1 s) for stepping up and down, respectively. When a subject was not able to maintain the required stepping rhythm, he was given a 30 s recovery period. Following the 30 s recovery he was required to recommence another set of the stepping cadence until fatigued and subsequently given another 30 s recovery period. This sequence was continued until complete exhaustion when even after a 30 s recovery the subject was not able to continue. The total number of sets and the stepping repetitions in each set were recorded for each subject (Table 1).
Sampling and hemostatic parameters Blood samples were collected before, immediately at fatigue, 1 and 24 h after the exercise. Blood (10 ml) was withdrawn by puncture from the antecubital vein and immediately collected in polypropylene tubes with anticoagulant and selected parameters measured using the following techniques. Platelet counts were performed with an electronic particle counter (Thrombocyte Analyser 147C-Analys Instrument) on whole blood kept in EDTA (1 mg/ml). Partial thromboplastin time (aPTT) and prothrombine time (PT) estimations were performed by citrate plasma analysis KC4 instrument (Amelunk, Germany) using appropriate reagents
166
J. Ribeiro et al.
Table 1
Mean value of repetitions number (Rep) performed in each one of the 21 sets of exercise protocol
Sets 1 Rep 172 N 10
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 21
58 10
73 10
52 10
47 10
41 10
41 10
31 9
31 8
26 8
35 7
27 6
20 6
18 5
18 4
18 4
16 4
12 3
13 1
7 1
4 1
The numbers of subjects (n) that executed each set are also presented.
Table 2 Means and standard deviations of hemostatic parameters observed at different moments of the experimental protocol Parameter studied
Time after exercise Before
Platelets (x109/l) vWF (U/l) aPTT (s) PT (s) TAT (g/l) Fibrinogen (g/l) FVIII (U/l) t-PA (g/l) PAI-1 (g/l) D-Dimer ((g/l) * **
276 0.89 32.7 12.3 1.9 2.8 1.16 3.6 28.2 95.9
± ± ± ± ± ± ± ± ± ±
0h 50 0.18 4.7 0.8 0.8 0.8 0.4 1.4 12.1 62.4
318 0.97 27.8 12.4 2.2 3.0 1.58 8.4 23.8 234.4
1h ± ± ± ± ± ± ± ± ± ±
50** 0.17** 4.3* 1.0 1.4 0.8 0.64* 5.1* 8.0* 262.2
283 0.89 30.0 12.6 3.4 2.8 1.33 3.3 19.6 137.8
24 h ± ± ± ± ± ± ± ± ± ±
45 0.17 4.1 1.0 4.7 0.8 0.36* 1.7 8.6* 116.5
303 0.87 33.0 12.5 2.5 2.8 1.04 3.7 26.5 153.5
± ± ± ± ± ± ± ± ± ±
46** 0.12 4.8 1.0 2.0 0.7 0.44 3.4 12.0 195.6
p < 0.05 vs. before. p < 0.01 vs. before.
(Diagnostic Grifolds SA). FVIII:C coagulant activity was assayed by the synthetic chromogenic substrate method using COATEST® Factor VIII (Chromogenix AB, Molndal, Sweden). von Willebrand factor (vWF) was measured using an enzyme-linked immunosorbent assay (ELISA), IMUBIND® vWF (American Diagnostica Inc., USA). Fibrinogen concentration was determined by the Clauss method with the respective reagents (Diagnostic Grifolds SA). For measuring thrombin-antithrombin complex (TAT) we developed an ELISA method using both capture antibody (TAT-EIA-C) and detecting antibody (TAT-EIA-D) from Affinity Biologicals Inc. (Enzyme Research Laboratories Inc., USA). D-dimer was evaluated by a solid phase enzyme immunoassay COALIZA® D-Dimer (Chromogenix AB, Molndal, Sweden). Plasminogen activator inhibitor (PAI-1) and tissue plasminogen activator (t-PA) were determined by IMUBIND® total t-PA and IMUBIND® Plasma PAI-1 ELISA kit (American Diagnostica Inc., USA), respectively.
Results
Statistical analysis
Discussion
Absolute values and percentages of variations based on pre-exercise values were expressed as means with standard deviations. Differences of means were tested with ANOVA for repeated measures, and the level of significance was set at ˛ < 0.05.
Exercise-dependent effects on the coagulant activity of the FVIII/vWF complex have been studied with various exercise protocols of varying intensities and durations. Despite these differences in experimental protocols, studies with healthy adults sug-
The mean number of exercise sets performed was 14.0 ± 5.0, and the total time of exercise was 1289 ± 294 s. The total number of sets, the mean number of repetitions in each set, and the number of subjects that performed every set are shown in Table 1. The absolute values for the studied hemostatic parameters are presented in Table 2, whilst the percentages of variation from the pre-exercise values are depicted in Fig. 1. Immediately after exercise, platelet counts, aPTT, FVIII, vWF and t-PA were significantly increased, while in contrast PAI-1 was significantly lower. At 1 and 24 h after the exercise, only FVIII and platelet counts, respectively, remained significantly elevated. At 1 h after exercise the PAI-1 was still significantly reduced.
Hemostatic response to acute physical exercise in healthy adolescents
167
Figure 1 Exercise effect on platelets, von Willebrand factor (vWF), activated partial thromboplastin time (aPTT), coagulation factor VIII (FVIII), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Values are expressed as means of percentage of variation from the values obtained before exercise (=100%).
gest significant increases in coagulant activity.2,3,14 Compared to adult experiments, the present study, with adolescent males also showed significant increase in FVIII/vWF concentrations immediately after exercise. In adult studies conducted at varying intensities, results have shown that FVIII activity can increase considerably, with concomitant variations of the vWF.5,9 In the present study however, despite the exhaustive exercise, the adolescent males demonstrated relatively lower enhancements in coagulant activity with FVIII increasing 36% and vWF increasing by 8.9%. These results concur with other studies that demonstrate FVIII increases are of greater magnitude when compared with those of vWF.5,6,9 Following exhaustive exercise in adults that consisted of dynamic or isometric contractions, the hypercoagulation response is also supported by the observed shortening of aPTT (7—38%).16 The PT response to exercise remains controversial, with several studies demonstrating both, significant shortening,9 and non-significant differences.1,3 The effect of acute exercise on plasma fibrinogen concentrations in adults is equivocal. Several studies demonstrated no differences,3,4 while others revealed significant increases17 or even significant decreases6 in plasma fibrinogen. The healthy adolescents of the present study produced a 15% shortening of aPTT immediately after exercise, while PT, TAT, and fibrinogen did not change. In contrast with adult responses in which the aPTT and PT changes seem to be persistent until 1 and 24 h after exercise, respectively,18 the present study revealed that aPTT returned to baseline after 1 h, and PT did not show any alteration.
The enhancement of FVIII and vWF, and the concomitant shortening of aPTT observed in this study suggest an activation of the coagulant system following exhaustive exercise. However, this activation seems to be less pronounced than observed in adult studies with exhaustive exercise.5,9 In addition, the response time of coagulant activity of adolescents appears to differ to that observed in adults. A plausible explanation for these responses can be related to differences in the ‘‘maturity status’’ of the responsible coagulant mechanisms, such as endothelial functionality. As the stimulus responsible for exercise-induced increases in plasma vWF and FVIII content seems to be mediated by -adrenergic receptors through a nitric oxide-dependent mechanism, the hemostastic system could be conditioned by endothelial function and be modified during the aging process.19 At rest, the aging-dependent endothelial dysfunction is usually associated with a basal thrombophilic state, i.e. an increased coagulation tendency with concomitantly decreased fibrinolytic activities.13 However, with physical exercise, an enhancement of fibrinolytic activity was observed, with increased plasmin formation resulting from the endothelial release of t-PA with a parallel decrease in PAI-1.3,4 Increases in fibrinolytic activities ranging from 75 to 250%, when compared to baseline levels, appear influenced by exercise intensity.5 In this study, the t-PA increased by 133% and PAI-1 decreased by 15.6% immediately after acute exercise and are consistent responses associated with a major activation of the fibrinolytic system in these healthy adolescents. Following short maximal exercise, increases in D-dimer con-
168 centrations (further marker of hyperfibrinolysis) have been measured in adults,1 but in this group of adolescents there was no significant increase in this parameter. The time to full post-exercise recovery to return to the resting levels of fibrinolytic parameters is equivocal, since several studies have registered 45—60 min after intense exercise,9 2 h after long distance running,2 or even 24 h after a marathon race.6 In our study, the t-PA returned to the resting levels 1 h after exercise while PAI-1 showed a significant decrease (30%) at 1 h post and was only normalized after 24 h. Previous studies in healthy adults and older subjects demonstrated that a single bout of exercise produced a pronounced increase in endogenous fibrinolysis, which may be protective against exertionrelated atherothrombotic events.20 However, an enhanced tendency for thrombosis may offset the increased fibrinolytic effect of exercise reported in adults.11 In this study, healthy adolescents showed a modest increase of coagulant activity and a more pronounced increase of fibrinolytic activity that might represent a powerful protection against thrombogenic events. Compared to adults, this difference in the thrombogenic response observed in adolescent males could be explained by the differing endothelial functionality associated with ageing. Whilst the results of the current study reveal desirable hemostatic responses observed in healthy adolescents, a logical extension of the current study would seem to question whether examining ‘‘well trained’’ adolescents might reflect the potential cumulative effects that both youth and fitness might have on beneficial hemostatic function.
Practical implications • This study provides normal adolescent values of hemostatic responses of adolescent males. • Developmentally, adolescent boys demonstrate no detrimental changes in their hemostatic system. • When compared to adults, the results reveal adolescents show different rates and ranges of coagulation and fibrinolysis parameters being activated by exhaustive exercise.
Acknowledgement The first author (JR) was kindly supported by CAPES, Brazil.
J. Ribeiro et al.
References 1. Molz AB, Heyduck B, Lill H, et al. The effect of different exercise intensities on the fibrinolytic system. Eur J Appl Physiol 1993;67:298—304. 2. Hansen JB, Wilsgard L, Olsen JO, et al. Formation and persistence of procoagulant and fibrinolytic activities in circulation after strenuous physical exercise. Thromb Haemost 1990;64:385—9. 3. El-Sayed MS, Lin X, Rattu AJ. Blood coagulation and fibrinolysis at rest and in response to maximal exercise before and after a physical conditioning programme. Blood Coagul Fibrinol 1995;6:747—52. 4. Rankinen T, Vaisanen S, Penttila I, et al. Acute dynamic exercise increases fibrinolytic activity. Thromb Haemost 1995;73:281—6. 5. Andrew M, Carter C, O’Brodovich H, et al. Increases in factor VIII complex and fibrinolytic activity are dependent on exercise intensity. J Appl Physiol 1986;60: 1917—22. 6. Prisco D, Paniccia R, Bandinelli B, et al. Evaluation of clotting and fibrinolytic activation after protracted physical exercise. Thromb Res 1998;89:73—8. 7. Womack CJ, Nagelkirk PR, Coughlin AM. Exercise-induced changes in coagulation and fibrinolysis in healthy populations and patients with cardiovascular disease. Sports Med 2003;33:795—807. 8. El-Sayed MS, El-Sayed Ali Z, Ahmadizad S. Exercise and training effects on blood haemostasis in health and disease: an update. Sports Med 2004;34:181—200. 9. Ferguson EW, Bernier LL, Banta GR, et al. Effects of exercise and conditioning on clotting and fibrinolytic activity in men. J Appl Physiol 1987;62:1416—21. 10. Matz RL, Andriantsitohaina R. Age-related endothelial dysfunction: potential implications for pharmacotherapy. Drugs Aging 2003;20:527—50. 11. Raitakari OT, Celermajer DS. Testing for endothelial dysfunction. Ann Med 2000;32:293—304. 12. Gallistl S, Sudi KM, Borkenstein M, et al. Determinants of haemostatic risk factors for coronary heart disease in obese children and adolescents. Int J Obes Relat Metab Disord 2000;24:1459—64. 13. van den Burg PJ, Hospers JE, van Vliet M, et al. Changes in haemostatic factors and activation products after exercise in healthy subjects with different ages. Thromb Haemost 1995;74:1457—64. 14. van den Burg PJ, Hospers JE, Mosterd WL, et al. Aging, physical conditioning, and exercise-induced changes in hemostatic factors and reaction products. J Appl Physiol 2000;88:1558—64. 15. Gallistl S, Sudi KM, Cvirn G, et al. Effects of shortterm energy restriction and physical training on haemostatic risk factors for coronary heart disease in obese children and adolescents. Int J Obes Relat Metab Disord 2001;25:529—32. 16. Hyers TM, Martin BJ, Pratt DS, et al. Enhanced thrombin and plasmin activity with exercise in man. J Appl Physiol 1980;48:821—5. 17. Torres-Guerra E, Diez-Ewald M, Vizcaino G, et al. Effect of stress electrocardiogram on platelet function, concentration of von Willebrand factor and fibrinogen in hypertensive patients and healthy subjects. Invest Clin 2000;41: 105—16. 18. Rocker L, Taenzer M, Drygas WK, et al. Effect of prolonged physical exercise on the fibrinolytic system. Eur J Appl Physiol 1990;60:478—81.
Hemostatic response to acute physical exercise in healthy adolescents 19. Jilma B, Dirnberger E, Eichler HG, et al. Partial blockade of nitric oxide synthase blunts the exercise-induced increase of von Willebrand factor antigen and of factor VIII in man. Thromb Haemost 1997;78:1268—71.
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20. Dufaux B, Order U, Liesen H. Effect of a short maximal physical exercise on coagulation, fibrinolysis, and complement system. Int J Sports Med 1991;12:S38—42.