Physical exercise has an effect on several clotting and fibrinolytic factors as well as blood platelet function (7). Interestingly, acute bouts of exercise-induced effects on hemostasis are different compared with effects induced by exercise training. The immediate physiological response to an acute bout of exercise is characterized by a transient hypercoagulable state with a shortened activated partial thromboplastin time and increased thrombin generation, elevated plasma levels of clotting factor VIII and von Willebrand factor, as well as an increase in platelet count and platelet reactivity (24,37). These reversible changes are more pronounced in individuals with a sedentary lifestyle than in those with a higher basal physical capability. In contrast, exercise training results in a down-regulation of the hemostatic potential indicated by a reduction in platelet reactivity and a reduction in the concentration and/or activity of various plasmatic clotting factors (21,38). The decline in hemostatic potential observed after exercise training is held responsible to contribute to a reduced risk of thrombotic cardiovascular events (40). So far, the mechanisms that are involved in these exercise-induced effects for both an acute bout of exercise and exercise training are not well understood.
Several recent studies have distinguished cell-derived microparticles (MP) released from leukocytes, erythrocytes, endothelial cells, and mainly from platelets as biological effectors that are involved in the regulation of vascular tone, coagulation, and inflammation (4,29). MP are strangulated plasma membrane fragments of activated or apoptotic cells with a size <1 μm. On their surface, most MP expose anionic phospholipids, various adhesion molecules, and/or coagulation factors, in particular, the tissue factor (TF) (23,25). These molecules enable them to alter vascular homeostasis and cellular processes. Various pathologies such as sepsis, cancer, diabetes mellitus, and cardiovascular disorders are associated with elevated levels of procoagulant MP (20,26). Beyond the total number, the composition of MP, especially the TF antigen level differing between healthy and disease status, is held responsible for triggering an MP-mediated thrombogenic effect (2). However, the fact that a substantial number of circulating procoagulant MP are also found in healthy individuals underline their role as an integral part for the maintenance of vascular homeostasis (1). Moreover, recent data have demonstrated some beneficial effects of MP including antithrombotic and anti-inflammatory properties as well as favorable influences on angiogenesis and vascular remodeling (10,30).
Recently, we have demonstrated that moderate endurance exercise increased the number of circulating platelet-derived MP (PMP) and enhanced the plasma procoagulant potential in healthy male volunteers (34). Physical inactivity is an established risk factor of morbidity and mortality (3). Thus, we were interested to investigate whether there are differences in the composition and procoagulant activity of circulating MP between individuals with a low (untrained) or a high (trained) physical performance. Furthermore, we examined the effect of a single bout of moderate endurance exercise on time-dependent changes in concentration and activity of circulating MP in both trained and untrained individuals.
MATERIALS AND METHODS
Sixteen nonsmoking healthy male volunteers with a mean age of 25 yr participated in the study. The physical health status of the participants was verified by recording the medical history and physical examination including electrocardiogram, blood pressure measurement, and main laboratory parameters (glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, creatinine, uric acid, fasting blood glucose level, hemogram, and erythrocyte sedimentation rate). Exclusion criteria were as follows: infection 4 wk before and during study enrollment/participation, individuals with drug abuse, smokers, or any ongoing medication. For standardization, participants were interdict to perform strenuous exercise outside the study protocol. The study protocol was approved by the ethics committee of the Friedrich Schiller University Jena, and written consensus was obtained from all participants.
Determination of physical performance and study cohorts.
About 1-2 wk before starting the singular endurance exercise test program, the peak oxygen uptake (V˙O2max) and individual anaerobic threshold (IAT) of each participant were determined by an incremental graded exercise on a bicycle ergometer. Starting at 50 W, exercise was stepwise increased by 50 W every 3 min until total exhaustion according to Stegmann et al. (35). For monitoring IAT, capillary blood samples were obtained from the previously hyperemized ear lobe at rest, at the end of each level of exercise (every 3 min), and at the end of the 1st, 3rd, 5th, and 10th min of recovery phase. Lactate concentrations were measured by the EBIO plus instrument (Eppendorf, Hamburg, Germany). V˙O2 was measured at 0.5-min intervals using an open spirometric system (Oxycon beta; Jaeger, Hoechberg, Germany). For discrimination of physical performance, volunteers were divided into the group of trained (relative V˙O2max >65 mL·min−1·kg−1) and untrained (relative V˙O2max ≤50 mL·min−1·kg−1) individuals (n = 8, each). These predetermined cutoff criteria were aligned according to previous studies for confirmation of comparability and standardization of physical exercise-triggered effects (11,13). According to these studies, the upper value of relative maximal oxygen consumption was chosen for reliable discrimination of individuals with availability of an efficient training status.
After 30 min of rest, participants fasting at the day of the test with free access to drinking water underwent a single standardized bout of endurance exercise on a bicycle ergometer with a constant power of 80% IAT for a period of 90 min. Definition of duration was aligned to previous studies, with extensive characterization of metabolic, hormonal, and immunologic responses (11,13). HR, blood pressure, and lactate were measured during exercise; data are given in Table 2.
Collection of blood samples.
Blood samples were taken before (rest), immediately after (post), 45 min after, and 2 h after exercise by an indwelling venous cannule during moderate stasis (40 mm Hg).
After discarding the first 3 mL of blood, we collected 9 mL each into two monovettes (Sarstedt, Nümbrecht, Germany) with lithium-heparin or EDTA as anticoagulants. After a short storage on ice, plasma samples were obtained by ad hoc centrifugation (3250g, 10 min at 4°C), and the plasma supernatant was kept at −80°C until analysis. Blood cell counts and hematocrit levels were measured in EDTA blood using a hematological analyzer (Act-Diff; Beckman Coulter, Brea, CA). Lactate concentration was measured by capillary blood samples collected from the previously hyperemized ear lobe as described above.
Characterization of MP by flow cytometry.
After thawing, 200 μL of plasma was mixed with 1 mL of Hank's buffered salt solution (without Ca2+/Mg2+), and the samples were centrifuged at 16,000g for 40 min at 4°C (32). After careful removal of 1 mL of supernatant, the remaining liquid containing MP was mixed with buffered saline solution (10 mmol·L−1 HEPES, 140 mmol·L−1 NaCl, 2.5 mmol·L−1 CaCl2, 1.5% bovine serum albumin, pH 7.4). MP were identified by flow cytometry according to their size and binding of 5 μL of fluorescein isothiocyanate (FITC)-labeled annexin V (Becton Dickinson, Heidelberg, Germany) as well as by the binding of PE-labeled antibodies against cell-specific antigens: anti-CD42a for PMP, anti-CD62E for endothelial cell-derived MP (EMP; 15 μg·mL−1 IgG1-PE; both Becton Dickinson) and anti-CD14 (15 μg·mL−1 IgG2a-PE; Sigma-Aldrich, St. Louis, MO) for monocyte-derived MP (MMP). Thereby the total amount of negatively charged particles exhibiting a phosphatidylserine-enriched surface for binding of coagulation factors was analyzed by annexin staining and discriminated from cell-derived MP by monitoring of specific, double-positive events to avoid gating of false-positive signals. In addition, MP were also stained with an FITC-labeled anti-TF antibody (20 μg·mL−1 IgG1-FITC; American Diagnostica, Stamford, CT). Concentration-matched isotype antibodies PE- or FITC-labeled IgG1 and IgG2a (Becton Dickinson) were used. After mixing 50 μL of the MP preparation with cell-specific antibodies, annexin V, or anti-TF, the samples were kept in the dark for 15 min at room temperature. Subsequently, all samples were mixed with 1 mL of buffered saline and 50 μL of counting beads with an established concentration close to 1000 beads per microliter (Flow-Count Fluorospheres; Beckman Coulter). The FACScan flow cytometer with CellQuest pro Software (Becton Dickinson) was set in logarithmic mode for forward and sideward scatters. Before MP analysis, a threshold for forward scatter was set by running latex beads with a size of 1.1 μm (Sigma-Aldrich), and only events below the threshold value were considered as events such as MP. Absolute numbers of MP were calculated by measuring a total number of 500 counting beads.
Procoagulant activity of MP.
To estimate procoagulant activities of MP, two different tests were performed: the ACTICHROME test (American Diagnostica), measuring the prothrombinase activity of FXa/Va of MP bound to an annexin V-coated surface in absence of any other plasma constituents; and a one-stage clotting test, monitoring the formation of a fibrin clot by measuring the optical density at 405 nm on a microplate reader in MP-containing plasma samples after recalcification and addition of a small amount of TF.
Data were tested for normal distribution and variance homogeneity using Kolmogorov-Smirnov and Levene tests, respectively, and were given as mean ± SD. Time- and group-dependent differences were tested for significances by two-way ANOVA for repeated measures using Bonferroni correction, followed by post hoc analyses using paired or unpaired Student's t-test. P ≤ 0.05 was considered to indicate statistical significance.
Anthropometric, performance, and control parameters.
Main anthropometric, physical performance parameters, and Borg RPE scale values are shown in Table 1. The higher cardiorespiratory capacity of trained subjects is indicated by the values of relative heart volume (13.4 ± 1.3 mL·kg−1), relative V˙O2max (68.1 ± 4.5 mL·min−1·kg−1), and maximum power (377.1 ± 58.2 W). Furthermore, physical performance as indicated by IAT was twice as high in trained subjects (285.8 ± 53.9 W) when compared with untrained (141.1 ± 54.0 W) individuals. Performance variables measured immediately after exercise reveal a trend of higher values for HR, systolic blood pressure, and plasma lactate in trained individuals. The given Borg RPE scale value was significantly higher in trained (15.5 ± 1.8) compared with untrained (13.5 ± 1.7) subjects (Table 1).
Hemodynamic and metabolic parameters during exercise.
As shown in Table 2, at rest, a lower HR and a higher systolic blood pressure were observed in trained compared with untrained individuals. The acute bout of exercise induced significant changes in HR, plasma lactate, and systolic and diastolic blood pressures in both groups (Table 2). Trained individuals exerted significantly higher values of systolic and lower values of diastolic blood pressure during as well as at the end of exercise. In the initial phase of exercise, the concentration of plasma lactate increased significantly in both groups, with a higher value in untrained subjects, and remained stable between 2.0 and 2.5 mmol·L−1 until the end of exercise test.
Hematogram and MP counts.
Physical exercise induced similar changes in hematocrit, leukocyte, and platelet counts in trained and untrained subjects (Table 3). Compared with baseline, elevated numbers of leukocytes were found for all time points with a peak at 2 h after exercise. Approximately the same was true for the changes in hematocrit. In contrast, platelet number was only increased immediately after exercise and returned to baseline values 45 min after exercise (Table 3).
No group-specific differences were observed in the numbers of annexin V-positive particles and MP subpopulations (PMP, MMP, EMP) before exercise (Table 3). After exercise, the number of annexin V-positive MP significantly increased above baseline levels with a maximum value at 45 min in trained subjects and at 2 h in untrained subjects. The difference between both groups was significant at 2 h (Table 3). A similar time course was found for PMP. Two hours after exercise, the number of PMP had nearly reached basal level in trained, whereas it was still significantly elevated in untrained subjects (Table 3). Interestingly, a transient increase in the numbers of EMP and MMP was only observed at 45 min after exercise in trained but not in untrained individuals (Table 3).
TF expression on MP.
The expression of TF protein on MP subpopulations at baseline and after exercise is shown in Figures 1A-C. At rest, the percentage of TF-positive PMP was 9.3% ± 5.4% in untrained and 5.1% ± 2.7% in trained subjects (Fig. 1A). After exercise, the concentration of TF-positive PMP decreased slightly immediately after but significantly 45 min after exercise to values around 50% of baseline levels in both groups. Two hours after exercise, counts of TF-positive PMP increased in trained but remained at a lower level compared with baseline in untrained individuals (Fig. 1A). The percentage of TF-positive EMP was almost similar between trained (31.2% ± 10%) and untrained (32.7% ± 9.4%) subjects (Fig. 1B). Exercise-induced enhanced values were found in trained individuals 45 min after the exercise test. With respect to MMP, the percentage of TF protein was significantly higher in untrained (32.4% ± 16.3%) compared with trained (18.2% ± 8.3%) individuals at baseline (Fig. 1C). Significant exercise-induced changes were found in untrained individuals, with an increase of TF-positive MMP up to 36.9% ± 14.7% 2 h after exercise.
Procoagulant activities of MP.
Prothrombinase activity of MP bound to annexin V-coated microplates was not different in trained and untrained at rest but was significantly enhanced after exercise in both trained (10.6 ± 2.3 nmol·L−1) and untrained (9.2 ± 2.8 nmol·L−1) subjects. This procoagulant activity of MP remained enhanced up to 2 h after exercise without any significant difference between trained and untrained subjects (Fig. 2).
The TF-initiated fibrin formation in MP-containing plasma samples was also increased after exercise by about 15% and remained higher compared with basal levels only in trained but not in untrained subjects (Fig. 3).
Our findings reveal that moderate endurance exercise triggers the release of cell-derived MP in healthy individuals. Cell-derived MP are known to promote coagulation owing to the surface exposure of negatively charged phospholipids and TF, the most potent activator of intravascular coagulation (25). Physical exercise has been shown to increase the hemostatic potential (15). Our data support the hypothesis that cell-derived MP significantly contribute to exercise-induced changes in hemostasis. Indeed, we observed an increase not only in the number of circulating annexin V-positive and changes in number of TF-positive MP but also in the prothrombinase activity of annexin V-positive MP. Furthermore, exercise also accelerated the TF-induced fibrin formation in MP-containing plasma samples.
Very recently, an article was published reporting that high shear stress applied in vitro of platelet-rich plasma samples increased thrombin generation and shedding of procoagulant PMP in healthy sedentary volunteers after strenuous exercise (5). These data provide further evidence that exercise could increase the capability of platelets to release procoagulant PMP and that PMP enhance the hemostatic potential. The data of Chen et al. (5) also support the hypothesis that shear stress is a major trigger of MP release during physical exercise. In line with that our data, some additional mechanistic aspects in vivo can be added. First, there is a trend to a higher amount and an increased activity of MP in trained subjects accompanied with higher values of systolic blood pressure compared with untrained individuals (Table 2). Second, we describe for the first time clear-cut differences in kinetic and dynamic of MP release and clearance: there is an almost continuous increase of MP amount in untrained subjects, reaching maximum values 2 h after exercise, whereas the peak value at a lower level in trained subjects is achieved at an earlier time point. Third, a similar behavior is observed with respect to PMP, according to the fact that PMP are the most abundant portions of all MP (16). Fourth, there are differences in the composition of MP with respect to TF exposure: MP of untrained individuals present higher amounts of surface-bound TF protein at baseline. However, this was only significant for MMP. And fifth, a more interesting finding is the strong decrease (around 50%) observed in the recovery phase with respect to TF-positive PMP, which may be caused by increased clearance in yet unknown mechanism, by the generation of PMP without abundant TF protein, or by consumption in unapparent coagulation processes. The latter one might contribute to a more preventive physiological adjustment during stress response. However, there is no information about TF activity under these circumstance because TF on MP is either in a latent (or "encrypted") form lacking coagulant activity or in an active (or "de-encrypted") form capable of initiating blood coagulation (4).
Detailed mechanisms of MP clearance from the circulation are unknown. It is discussed that MP could be cleared from the circulation by phospholipases, by direct mechanisms such as phosphatidylserine exposure and subsequent phagocytosis, or by indirect mechanisms such as opsonization protein S and complement protein (9,39).
Beyond high shear rates, many potent agonists have been identified to stimulate MP formation and shedding in vitro and ex vivo. However, in vivo mechanisms of MP generation are mainly unknown. On the basis of in vitro experiments, potent stimuli for the release of MP with varying potency are epinephrine < ADP < thrombin < collagen < calcium ionophor (A23187) (16). Many of these agonists are influenced by phys ical exercise and might trigger the stimulation of blood cells and the release of procoagulant MP (14,15,33). It is well known that a single bout of physical exercise induces a marked increase in plasma catecholamine concentration especially norepinephrine (18). Furthermore, platelets of trained subjects exhibit less sensitivity against norepinephrine owing to down-regulation of α2-adrenoceptor molecules (22) and training decreases exercise-induced plasma catecholamine levels (27). In our study, these hormone-mediated effects were not proven in detail and might be overcome by shear stress-induced MP generation.
Despite identical IAT-guided exercise protocol in trained and untrained individuals, the Borg RPE scale value was significantly higher in trained (15.5 ± 1.8) compared with untrained (13.5 ± 1.7) subjects. We also found higher values of blood pressure in trained individuals. Both parameters indicate that, in trained individuals, the cardiopulmonary exertion was raised, causing a moderate bias comparing both groups. However, because of the author's perception, only an IAT-guided exercise bout is a reliable and established method to elaborate variations between trained and untrained subjects. In line with increased values of systolic blood pressure, the increased number of MP in trained individuals might be caused by enhanced shear stress in these conditions. Overall, the principal cause of pronounced generation of MP in trained subjects remains unclear because trained participants were characterized by an increased workload represented by hemodynamic responses and/or RPE ratings. However, we clearly observed variations in clearance rate of circulating MP in between the groups, suggesting differences in the biological effectiveness of these stress-induced mediators, which was the main objective of our study. For detailed elucidation of possible (patho-) physiological mechanisms responsible for increased MP release, an enlarged study cohort might be analyzed in an exercise protocol, which is either not IAT-guided or monitored hemodynamic variables should be analyzed as covariates during data manipulation.
Physical exercise, in particular, strenuous exercise, is known to be associated with increased plasma levels of proinflammatory cytokines and other surrogates of a mild systemic inflammatory response (11,12,31). However, the underlying mechanisms of the exercise-induced systemic inflammation are not well understood. Systemic inflammation is frequently associated with increased levels of circulating MP, and there is increasing evidence that MP, including PMP, may play a crucial role in the pathogenesis of systemic inflammatory reaction (29,36). Because of the surface expression of various integrins and selectins, PMP are capable of adhering to leukocytes and endothelial cells. Such interactions have been shown to modulate target cells to an inflammatory and/or a proliferating phenotype (6,8,32). Thus, one may speculate that formation of PMP may be involved in the development of systemic inflammatory reaction during and after physical exercise. Because of their procoagulant and proinflammatory activities, the role of circulating MP is often seen as bad it may have also some beneficial effects (10). MP are also involved in the processes of vasculogenesis and angiogenesis. They recruit progenitor cells to the site of the vessel wall and stimulate endothelial cells and smooth muscle cells to release growth factors, e.g., vascular endothelial growth factor, platelet-derived growth factor, basic fibroblast growth factor, as well as reactive oxygen species, which are required for cell differentiation and migration (17,19,28). Therefore, MP could be important for vessel repair and wound healing. To maintain the homeostatic equilibrium released, MP are quickly incorporated and digested by phagocytes. Thus, in conditions with an imbalance of the release and elimination of MP, possibly induced by strenuous exercise or insufficient time of physical regeneration, higher levels of circulating MP could be harmful regarding their procoagulatory and proinflammatory potential.
Numerous epidemiological and prospective cohort studies confirm an important and beneficial role of moderate endurance exercise for the prevention and treatment of cardiovascular diseases (7,40). Thus, MP that are released into the circulation by moderate endurance exercise as shown in the present study may play a beneficial role in terms of adaptation to procoagulant and proinflammatory stress. Another mechanism of adaptation might be a more expeditious clearance of MP from the circulation. However, further studies with larger cohorts are needed to clarify the exact role of exercise-induced MP release in physiological and pathophysiological conditions.
The authors did not receive any financial support for this work and have no competing interests relevant to the article.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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