In healthy individuals, physical exercise induces an activation of blood coagulation resulting in thrombin formation, as indicated by increased plasma levels of thrombin-antithrombin III (TAT) complexes, which is not always accompanied by fibrin formation (4, 17). Unchanged plasma levels of fibrinopeptide A (FPA) after short and/or submaximal exercise(17,31) and slightly increased FPA concentrations reported after a 2-h triathlon (6) and after a marathon race (25) suggest that minimal exercise-induced fibrin generation only occurs during prolonged strenuous exercise.
An exercise-induced increase of fibrinolytic activity in vitro has been reported consistently(4,6,8,18,20,26) and has been linked to the release of tissue plasminogen activator (t-PA) by the vascular endothelium (8,20). It remains, however, a matter of debate whether increased in vitro fibrinolytic activity reflects effective fibrin(ogen)olysis in vivo (20). Moderate elevations of fibrin degradation products were observed after prolonged(3,4,6,14,18,19,26) but not after short-term strenuous exercise(5,9,20,31). The discrepancies with respect to in vivo fibrinolysis might be explained by the lack of fibrin formation as the substrate(4,5,17,20,31). This interpretation of available data supports the hypothesis that the hemostatic system is well balanced during exercise with in vivo fibrinolysis counteracting in vivo fibrin generation.
Based on these data, we hypothesized that moderate exercise would activate predominantly the fibrinolytic system, while very heavy exercise in addition to plasmin formation would also give rise to thrombin and fibrin formation. The relationship between exercise intensity and activation of coagulation and fibrinolysis has, however, never been studied systematically using neoantigens indicative of in vivo activity. Therefore, we determined molecular markers of platelet activation (β-thromboglobulin, (BTG)), of thrombin(prothrombin fragment 1+2 (PTF 1+2), TAT), of fibrin (FPA) and of plasmin formation (plasmin-α-antiplasmin (PAP) complexes) as well as markers of the fibrinolytic activity (fibrin degradation products (FbDP)) in healthy trained young men before, during, and after 1 h of running at moderate and very heavy intensity.
SUBJECTS AND METHODS
Twelve young men (mean age 24 yr; range 21-35 yr) without evidence of hemostatic disorders (protein C, 71% to >100%; protein S, 73% to >100%; antithrombin III, 82% to >100%; prolongation of aPTT by activated protein C, 2.9- to 3.2-fold) were recruited for the study. Participants were all engaged in regular noncompetitive exercise and considered to be well trained. All subjects were nonsmokers and had been without medication for at least 2 mo. Anthropometric data are summarized in Table 1. All participants gave their written informed consent before entering the study.
Incremental graded exercise testing. Subjects performed an incremental graded exercise test on a treadmill starting at an initial velocity of 8 km·h-1 with increments of 2 km·h-1 every 3 min until exhaustion. An ECG was recorded continuously, and a representative sample was printed out before each increment and within the last seconds of exercise to assess the maximal heart rate. Maximal oxygen uptake (˙VO2max) was measured by means of a metabolic cart(Oxycongamma, Mijnhardt b.v., Bunnik, The Netherlands) applying an open circuit method. Capillary blood samples were obtained from the fingertip for measurement of plasma lactate at rest, at the end of each level of exercise, and in the 1st, 3rd, 5th, and 10th minute of the recovery period. The individual anaerobic threshold (IAT) was determined applying the method of Stegmann et al. (29). Briefly, IAT is determined from the blood lactate concentration curve by means of a tangent.
1-h exercise tests. Three and 5 d after determination of˙VO2max, subjects ran on a treadmill for 1 h, either at a constant velocity corresponding to 80% IAT (68 ± 4% ˙VO2max) or at 100% IAT (83 ± 5% ˙VO2max). The sequence of the low and high intensity running was random. All tests were carried out between 4 p.m. and 6 p.m. Heart rate was recorded continuously by a sport tester (Polar Electro, Kempele, Finland), and plasma lactate was measured every 15 min during exercise to calculate a mean plasma lactate concentration. Mean heart rate during high intensity running was 94 ± 4% of maximum heart rate measured at the ˙VO2max test and 82 ± 5% during the low intensity running.
According to the recommendations of the American College of Sports Medicine(1) for nonathletic adults, classification of the exercise intensities based on percentages of ˙VO2max indicates that the low and high intensity to which our subjects were exposed correspond to“moderate” and “heavy” to “very heavy” exercise, respectively. The mean heart rate during exercise reflects, however,“moderate” to “heavy” and “very heavy” exercise intensity, respectively. Considering that our subjects were well trained and that well-trained men have a 2% higher heart rate than untrained men (30) at comparable% ˙VO2max we regard the lower exercise intensity as “moderate” and the higher intensity as“very heavy”. This terminology is in accordance with usual training recommendations based on IAT for athletes; velocities corresponding to 80% of IAT characterize “regenerative” and those of 100% of IAT characterize “intense” endurance training.
Blood sampling and laboratory methods
Blood samples were drawn by a clean venipuncture (20 gauge needle) from an antecubital vein under controlled venous stasis of 45 torr using the Sarstedt system (Sarstedt Nümbrecht, Germany) at rest, after 30 min of exercise, immediately after exercise, and after 1 h recovery. Blood samples were collected before exercise and after recovery following a rest period of 30 min in supine position.
Samples were collected in the following sequence. 1.) 4.5 mL of blood was added to 0.5 mL of CTAD-PPACK anti-coagulant (stock solution containing 25 mL of citrate-theophylline-adenosine-dipyridamole (Becton Dickinson, Rutherford, NJ) plus 5 mg of phenyl prolyl arginine-chloromethylketone (Calbiochem, La Jolla, CA) giving a PPACK concentration of 382 μM) for measurement ofβ-thromboglobulin (BTG, radioimmunoassay supplied by Amersham, Buckinghamshire, UK), prothrombin fragment 1 + 2 (Enzygnost-F1 + 2, Behring, Marburg, FRG), thrombin-antithrombin III complexes (Enzygnost-TAT, Behring), and fibrinopeptide A (FPA, radioimmuno reagents supplied by Imco, Stockholm, Sweden). The procedures of the latter assay have been reported in detail previously (17). 2.) 9 mL of blood added to 1 mL of 0.106 M trisodium citrate for assessment of activated partial thromboplastin time (aPTT) with use of Pathromtin (Behring, Marburg, FRG), of tissue plasminogen activator antigen (t-PA) (TintElize tPA, Biopool, Umea, Sweden), of plasmin-antiplasmin (PAP) complexes by radioimmunoassay (Enzygnost PAP, Behring, Marburg), and of fibrin degradation products (FbDP, Fibrinostika, Organon, Teknika, Turnhout, Belgium). PAP complexes are expressed in nmol·L-1 assuming a molecular weight of 140 kDa. 3.) 4.5 mL of blood added to 7.5 mg of dry EDTA for leukocyte and platelet count, hemoglobin determination (Coulter Counter, Coulter Electronics), and measurement of hematocrit (micro-hematocrit centrifuge, Hettich, Tuttlingen, FRG) in duplicate after 6 min of 13,000g.
Immediately after blood sampling tubes 1 and 2 were rapidly put into melting crushed ice for 10 min and thereafter centrifuged at 4 °C for 30 min at 2000 × g. Multiple aliquots of plasma were snap-frozen in liquid nitrogen and stored at -80 °C until analysis. Changes in plasma volume were calculated according to the method of Dill and Costill(13). The results for platelets and for proteins with a molecular weight >30,000 (16), i.e., all except for BTG and FPA were corrected for changes of plasma volume (PV) occurring during exercise by the following factor: (100+ΔPV)/100, where ΔPV is the change of PV given in percent.
All samples obtained from the same subjects were analyzed in one particular run of the respective immuno-assays. Intra-assay coefficients of variation(CV) are below 5% for t-PA antigen and PAP complexes; below 7% for BTG, TAT, and FPA; and below 10% for PTF1+2. Interassay CV does not exceed intra-assay CV except for BTG (10-15%).
Except for PAP, normal values were established using the same assays in 24 samples taken from resting healthy male subjects of a comparable age group(20-30 yr) between 2 and 3 p.m. at least 2 h after a light meal. The upper limit of normal values was obtained by adding 2 SD to the mean value. Reference values for PAP were taken from the product information of the supplier.
Data were analyzed separately for each exercise test by ANOVA with repeated measures for main effect of exercise. When ANOVA revealed a significant effect, Student's t-test was used for post hoc testing to examine the difference between values at baseline and those obtained during or after exercise. For these three multiple comparisons, a Bonferroni adjustment was made multiplying a particular P value by 3. The level of statistical significance was set at P < 0.05 (two-sided). Results are reported as mean ± SE unless otherwise stated.
Characteristics of exercise testing are summarized inTable 1. The exercise intensities classified as moderate and very heavy are well documented by the differences in% ˙VO2max, mean heart rate, mean plasma lactate, and in the decrease of PV.
Platelet count increased significantly to similar levels with both forms of exercise (Table 2). During moderate exercise, BTG increased slightly with a significant rise after 30 min while plasma levels of BTG doubled after 60 min of very heavy exercise. There was a significant shortening of aPTT compared with baseline in all examinations with a considerably more pronounced response after very heavy exercise(Table 2). Regarding neoantigens, moderate exercise induced only slight and (except for TAT in the recovery phase) insignificant increases of PTF 1+2, TAT, and FPA (Fig. 1). Following very heavy exercise, significant elevations of all these parameters were recorded. Changes, however, occurred all within the range of normal values.
We observed a marked activation of the fibrinolytic system after moderate exercise as indicated by t-PA antigen levels and PAP concentrations rising on the average three- and two-fold, respectively (Fig. 2). There was, however, only a 20% increase of FbDP, reaching statistical significance in the recovery phase only. In response to very heavy exercise, t-PA antigen and PAP rose in controls by five- and three-fold, respectively, while concentrations of FbDP increased significantly by 50% during recovery.
This study examined the effects of two different exercise intensities on molecular markers of thrombin, fibrin, and plasmin generation in healthy young trained men. It demonstrates that a moderate intensity leads predominantly to increased plasma levels of PAP complexes while very heavy exercise, in addition to plasmin formation, gives rise to significant increases in plasma levels of PTF1+2, TAT, and FPA. We conclude that the activation of fibrinolysis occurs at a lower level of exercise intensity than activation of coagulation. Furthermore, in response to very heavy exercise the fibrinolytic system seems to be activated to the higher degree than hemostasis, suggesting that fibrinolytic activation counteracts exercise-induced thrombin and fibrin formation.
The finding of increased thrombin and fibrin formation after prolonged very heavy exercise is in accordance with data reported after marathon running(25) and a triathlon competition(6). Moderate exercise of untrained subjects over 1 h resulting in a mean heart rate of 146 ± 9 beats·min-1 (17) caused a mild but significant increase of plasma levels of PTF1+2 and TAT complexes. No significant changes of these parameters, however, were observed in our group of endurance-trained individuals of comparable age and at a comparable exercise intensity. This observation supports the notion that physical conditioning may reduce the exercise-induced activation of coagulation at submaximal levels as has been reported with regard to coagulation times by Ferguson and Guest(15). In previous investigations of various groups, plasma levels of FPA were not increased after long-term moderate exercise(17) nor after stepwise maximal exercise leading to exhaustion within about 15 min (14,20,31). Taken together, these studies suggest that only prolonged very heavy exercise leads to a minor but significantly increased fibrin formation in healthy young individuals.
The exercise-induced release of t-PA antigen depends on exercise intensity as was also shown by various groups(8,18,24). Considering that measurement of t-PA, antigen does not necessarily reflect the activity of t-PA, significant increases of PAP complexes indicate an enhanced in vivo plasmin formation after exercise at both intensity levels. Our data on markers ofin vivo activity of coagulation and fibrinolysis are consistent with previous studies assessing in vitro activity by coagulation and clot lysis time (2,12,15), implying that activation of fibrinolysis occurs at lower levels of exercise than activation of coagulation.
The effectiveness of exercise-induced fibrinolytic activation has been questioned (14,20), since high increases of t-PA were not accompanied by corresponding changes of fibrin degradation products after short (20) or prolonged strenuous exercise(14). The presented data demonstrate a greatly enhanced plasmin formation as indicated by severalfold increases of PAP complexes in response to exercise. This activation of fibrinolysis resulted in a significant increase of FbDP in the presence of increased plasma levels of FPA with very heavy exercise while moderate exercise not significantly affecting FPA levels did not induce an increase of FbDP. Thus, our data are compatible with the notion that unchanged fibrin degradation products in the presence of exercise-induced increases of t-PA and PAP complexes reflect the lack of substrate (i.e., fibrin) rather than an ineffective fibrinolysis.
A comparison of the magnitude of exercise-induced thrombin, fibrin, and plasmin formation is difficult even when changes are expressed in molar concentrations 1) because not all plasmin is bound by α-2-antiplasmin or thrombin by antithrombin III and 2) because of differences in plasma half-lifes. Half-lifes were estimated to be a few minutes for TAT complexes(27) and FPA (22), 90 min for PTF 1+2 (7), and 12 h for PAP complexes(11). Considering the above stated limitations, the increase of PTF1+2 in the order of 0.13 nmol·L-1 and of PAP complexes in the order of 4.7 nmol·L-1 after very heavy exercise suggests nevertheless a disproportionally greater activation of fibrinolysis than of coagulation.
Our findings that moderate exercise leads predominantly to plasmin formation and that marked plasmin formation counteracts minor fibrin formation after very heavy exercise imply that young trained healthy male subjects are not at an increased risk of thrombosis during exercise. It is conceivable, however, that especially in older subjects with preexisting arteriosclerotic lesions the activation of coagulation during heavy exercise may facilitate the occurrence of thrombotic occlusion even though fibrinolysis is activated simultaneously. This notion fits well with the observation that, especially in untrained, older individuals, vigorous exercise is associated with an increased risk of sudden death (28) and myocardial infarction (21,32). Predominant and repeated activation of fibrinolysis associated with moderate exercise may, however, counteract fibrin deposition involved in the pathogenesis of atherosclerosis(10), and it may thereby contribute to the benefit of physical exercise in retarding the arteriosclerotic process as it is well documented in patients with coronary artery disease(23).
The authors thank R. Dussen, G. Schmeiser, and E. Kohl for skillful technical assistance and V. Swonke for secretarial work.
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Keywords:©1998The American College of Sports Medicine
HEMOSTASIS; THROMBIN; FIBRIN; FIBRINOPEPTIDE A; PLASMIN; TISSUE PLASMINOGEN ACTIVATOR; FIBRIN DEGRADATION PRODUCTS