Acute hypoxic exposure increases vascular thrombotic risk. The release of procoagulant-rich microparticles from neutrophils accelerates the pathogenesis of inflammatory thrombosis. The present study explicates the manner in which interval and continuous exercise regimens affect neutrophil-derived microparticle (NDMP) formation and neutrophil/NDMP-mediated thrombin generation (TG) under hypoxic condition. A total of 60 sedentary males were randomized to perform either aerobic interval training [AIT; 3-min intervals at 40% and 80% V̇O2max (maximal O2 consumption)] or moderate continuous training (MCT; sustained 60% V̇O2max) for 30 min/day, 5 days/week for 5 weeks, or to a control (CTL) group who did not receive any form of training. At rest and immediately after hypoxic exercise test (HE, 100 W under 12% O2 for 30 min), the NDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. Before the intervention, HE (i) elevated coagulant factor VIII/fibrinogen concentrations and shortened activated partial thromboplastin time (aPTT), (ii) increased total and tissue factor (TF)-rich/phosphatidylserine (PS)-exposed NDMP counts and (iii) enhanced the peak height and rate of TG promoted by neutrophils/NDMPs. Following the 5-week intervention, AIT exhibited higher enhancement of V̇O2max than did MCT. Notably, both MCT and AIT attenuated the extents of HE-induced coagulant factor VIII/fibrinogen elevations and aPTT shortening. Furthermore, the two exercise regimens significantly decreased TF-rich/PS-exposed NDMP formation and depressed neutrophil/NDMP-mediated dynamic TG at rest and following HE. Hence, we conclude that AIT is superior to MCT for enhancing aerobic capacity. Moreover, either AIT or MCT effectively ameliorates neutrophil/NDMP-promoted TG by down-regulating expression of procoagulant factors during HE, which may reduce thrombotic risk evoked by hypoxia. Moreover, either AIT or MCT effectively ameliorates neutrophil/NDMP-promoted TG by down-regulating expression of procoagulant factors during HE, which may reduce thrombotic risk evoked by hypoxia.

CLINICAL PERSPECTIVES

  • AIT exhibits a larger cardiopulmonary adaptation than does MCT.

  • An acute bout of 12% O2 exercise (HE) may accelerate endogenous TG in NRP via increasing levels of procoagulant neutrophils and NDMPs under inflammatory conditions.

  • Either AIT or MCT effectively ameliorates neutrophil/NDMP-promoted TG by down-regulating expression of procoagulant factors during HE, which may reduce thrombotic risk evoked by hypoxia.

INTRODUCTION

Acute exposure to a hypoxic environment, such as high altitude, has been suggested to increase the risk of venous thromboembolism [1,2]. Pathological investigations have also demonstrated that a hypercoagulable state contributes to increased risks of vascular thrombotic events in patients with episodic hypoxia, such as obstructive sleep apnoea and chronic obstructive pulmonary disease [3,4]. High-intensity exercise may elicit greater cardiopulmonary adaptations than low and moderate levels of exercise [5]. However, physical exercise imposes, paradoxically, both enhancing and suppressing effects on the haemostatic system, depending on the type and intensity of exercise [6,7]. To the best of our knowledge, what kind of exercise strategy improves aerobic capacity and simultaneously increases the resistance to thrombotic risk provoked by hypoxia has not yet been established.

Neutrophil and thrombocyte co-localization to the wall of damaged or stimulated blood vessels is an essential component of a multistep cascade in inflammatory thrombosis [8,9]. Moreover, the procoagulant microparticles released from activated neutrophils substantially contribute to the thrombotic occlusion of blood vessels [10]. The negatively charged phosphatidylserine (PS) exposed on the microparticles can bind coagulation factors VIII (FVIII), FVa and FXa, providing a strongly catalytic surface for the assembly of prothrombinase and tenase [11,12]. Additionally, tissue factor (TF) acts as a receptor for the FVII/VIIa and the formation of this complex facilitates the cleavage of FX to FXa [13]. Activated prothrombinase complex catalyses the generation of thrombin from prothrombin, resulting in the formation of a fibrin clot [1113]. Acute hypoxic exposure may affect endogenous thrombin generation (TG) by modulating the production of FVIII in a concentration-dependent manner [2]. However, no clear and comprehensive pictures of the distinct effects of various exercise regimens on neutrophil-derived microparticle (NDMP) formation and neutrophil/NDMP-mediated TG under hypoxic condition are available.

A prior bout of moderate-intensity exercise [MIE; 40% V̇O2max (maximal oxygen consumption)] has been observed to reduce the risk of inflammatory thrombosis evoked by vigorous exercise (80% V̇O2max), this effect being a form of pre-conditioning [14]. Recently, our clinical investigation in the healthy or heart failure (HF) population further revealed that aerobic interval training (AIT) that consists of alternating low- (40% V̇O2max) and high-(80% V̇O2max) intensity exercise effectively suppressed oxidative stress/inflammation associated with haemodynamic/haemorheological [15,16] or immune [17] dysfunction. Accordingly, we further hypothesize that AIT effectively reduces neutrophil/NDMP-promoted TG by down-regulating expression of procoagulant factors under hypoxic condition.

To test the hypotheses, the effects of interval and continuous exercise regimens on (i) intrinsic and extrinsic coagulation systems in blood, (ii) total and procoagulant NDMP formation and (iii) dynamic TG mediated by neutrophils and NDMPs under hypoxic condition were explored. The aim of the present study was to establish an effective exercise strategy for improving individual aerobic capacity and simultaneously ameliorating the risk of inflammatory thrombosis associated with hypoxic stress.

MATERIALS AND METHODS

Subjects

A total of 60 sedentary males who were non-smokers, non-users of alcohol/medications/vitamins and cardiovascular/haematological risk-free were recruited in 1 year from Chang Gung University, Taiwan. None of the subjects had regular exercise habits (i.e. exercise frequency once per week, duration<20 min) or were exposed to high altitudes (> 3000 min) for at least 1 year before the experiment. These subjects were randomly divided into three groups: AIT (n=20), moderate continuous training (MCT, n=20) and control (CTL, n=20) groups. Anthropometric and cardiovascular characteristics of all individuals did not differ significantly before interventions (Table 1). All subjects arrived at the testing centre at 09:00 h to eliminate any possible diurnal effect. Participants were instructed to fast for at least 8 h and to refrain from strenuous physical exercise for at least 48 h before sampling. The investigation followed the Declaration of Helsinki and was approved by the Chang Gung Memorial Hospital Institutional Review Board, Taiwan. All subjects provided informed consent after the experimental procedures were explained.

Table 1
The effects of various exercise regimens on cardiopulmonary fitness

Values are means±S.E.M. +P<0.05, Pre compared with Post; #P<0.05, AIT compared with MCT. BMI, body mass index; BP, blood pressure; Post, after the intervention; Pre, before the intervention.

AITMCTCTL
ParameterPrePostPrePostPrePost
Anthropometric 
 Age (years) 23.2±1 – 23.1±1 – 23.3±2 – 
 Weight (kg) 64.1±2.4 63.0±2.2 65.4±2.5 65.4±2.7 65.0±2.4 65.6±2.1 
 Height (cm) 171.2±1.3 171.2±1.3 172.1±1.9 172.1±1.9 171.0±2.0 171.0±2.0 
 BMI (kg/m221.9±0.8 21.5±0.7 22.1±0.7 22.1±0.7 22.2±0.6 22.4±0.6 
 HR (beats/min) 72±1 68±2+ 73±2 69±2+ 72+2 71+3 
 Systolic BP (mmHg) 118±3 113±3+ 116±4 114+3+ 117±3 116±3 
 Diastolic BP (mmHg) 74±2 72±3 74±3 72±2 75±4 74±3 
Peak exercise performance 
 Work rate (W) 188±8 244±9+# 182±7 220±9+ 190±8 198±9 
 Exercise time (min) 24±1 31±1+# 23±1 28±1+ 24±1 25±1 
 HR (beats/min) 196±2 198±2 194±4 199±3 190±3 197±2 
V̇E (l/min) 111±2 128±3+# 107±2 123±1+ 112±3 115±3 
V̇O2 (ml/min per kg) 42.4±0.4 54.8±0.8+# 42.1±0.2 47.0±0.1+ 43.8±1.6 45.0±2.8 
V̇CO2 (ml/min per kg) 51.4±0.1 65.5±1.4+# 51.1±0.4 56.1±0.3+ 52.5±1.9 54.1±2.1 
AITMCTCTL
ParameterPrePostPrePostPrePost
Anthropometric 
 Age (years) 23.2±1 – 23.1±1 – 23.3±2 – 
 Weight (kg) 64.1±2.4 63.0±2.2 65.4±2.5 65.4±2.7 65.0±2.4 65.6±2.1 
 Height (cm) 171.2±1.3 171.2±1.3 172.1±1.9 172.1±1.9 171.0±2.0 171.0±2.0 
 BMI (kg/m221.9±0.8 21.5±0.7 22.1±0.7 22.1±0.7 22.2±0.6 22.4±0.6 
 HR (beats/min) 72±1 68±2+ 73±2 69±2+ 72+2 71+3 
 Systolic BP (mmHg) 118±3 113±3+ 116±4 114+3+ 117±3 116±3 
 Diastolic BP (mmHg) 74±2 72±3 74±3 72±2 75±4 74±3 
Peak exercise performance 
 Work rate (W) 188±8 244±9+# 182±7 220±9+ 190±8 198±9 
 Exercise time (min) 24±1 31±1+# 23±1 28±1+ 24±1 25±1 
 HR (beats/min) 196±2 198±2 194±4 199±3 190±3 197±2 
V̇E (l/min) 111±2 128±3+# 107±2 123±1+ 112±3 115±3 
V̇O2 (ml/min per kg) 42.4±0.4 54.8±0.8+# 42.1±0.2 47.0±0.1+ 43.8±1.6 45.0±2.8 
V̇CO2 (ml/min per kg) 51.4±0.1 65.5±1.4+# 51.1±0.4 56.1±0.3+ 52.5±1.9 54.1±2.1 

Training protocols

Both AIT and MCT groups performed exercise regimens on a stationary bicycle ergometer five times a week for 5 weeks [1517]. For comparison purpose, CTL participants did not perform any exercise but were carefully monitored and information of physical activity and healthy eating was recorded.

AIT subjects warmed-up for 3-min at 30% of V̇O2max before continuing five exercise cycles, 3-min each at 80% of V̇O2max alternated with a 3-min active recovery at 40% of V̇O2max. The exercise session was terminated by a 3-min cool-down at 30% of V̇O2max. The MCT group had the same warm-up and cool-down protocols as the AIT group except for the training period which was 30 min at 60% of V̇O2max [1517]. Each subject used a heart rate (HR) monitor (Tango, SunTech Medical) to obtain the assigned intensity of exercise. Borg 6-to-20 scale was used to assess the rate of perceived exertion during each 3-min testing and after each exercise session. The work-rate of the bicycle ergometer was adjusted continuously to ensure that the intensity of exercise matched the target HR throughout the training period. The two exercise protocols were isovolumic at the same exercise duration [i.e. AIT exercise volume: 6 min (40% V̇O2max+80% V̇O2max × 5 cycles=MCT exercise volume: 30 min (60% V̇O2max)]. The CTL group kept to their original diets and physical activity habits for 5 weeks.

All subjects recorded their daily activities and nutrition intakes with questionnaires throughout the experiment. Participants were instructed to refrain from extra regular exercise until the end of the present study. Moreover, their compliance rates to the three interventions were 100%.

Graded exercise tests

Each subject performed a graded exercise test (GXT) on a bicycle ergometer (Corival 400, Lode) to assess their aerobic fitness and haemodynamic functions 4 days before and 4 days after the 5-week interventions [17]. The GXT comprised 2 min of unloaded pedalling followed by a continuous increase in work-rate of 20–30 W per 3 min until exhaustion (i.e. V̇O2max). Minute ventilation (V̇E), V̇O2 and carbon dioxide production (V̇CO2) were measured breath by breath using a computer-based system (MasterScreen CPX, Cardinal Health). Mean arterial pressure (MAP) was measured using an automatic blood pressure system (Tango, SunTech Medical) and arterial O2 saturation was monitored by finger pulse-oximetry (model 9500, Nonin Onyx) [17]. The V̇O2max was defined by the following criteria: (i) the level of V̇O2 increased less than 2 ml/kg per min over at least 2 min; (ii) HR exceeded its predicted maximum; (iii) the respiratory exchange ratio exceeded 1.2 times; and (iv) the venous lactate concentration exceeded 8 mM, consistent with the guidelines of American College of Sports Medicine for exercise testing [18].

Hypoxic exercise tests and blood collection

Each subject performed the hypoxic exercise (HE) test 2 days before the intervention and 2 days after the intervention in an air-conditioned normobaric hypoxia chamber (Colorado Mountain Room), as mentioned in our previous studies [17,19]. The hypoxia chamber was maintained at a temperature of 22±0.5°C with a relative humidity of 60±5%; a CO2 scrubber eliminated CO2 in the air (<3500 ppm) [17,19]. The HE test on the bicycle ergometer required 50 W of warm-up for 3 min, an increase in work rate to 100 W of continuous exercise for 30 min and then recovery to 50 W of cool-down for 3 min. During the test, the O2 concentration was set to 12%, which corresponds to altitudes of 4460 m.

At rest and immediately after the HE test, 30 ml blood samples were collected from an antecubital vein using a clean venipuncture (20 gauge needle) under controlled venous stasis at 40 torr. The first 2 ml of blood was discarded and the remaining blood was used to measure haematological parameters. Blood cells were counted using a Sysmax SF-3000 cell counter (GMI) [17].

Intrinsic and extrinsic coagulation systems

The activated partial thromboplastin time (aPPT, intrinsic coagulation time), prothrombin time (PT, extrinsic coagulation time), FV, FVII, FVIII, protein C and fibrinogen in plasma were measured by commercial kits (Technoclone). All blood samples were processed according to instructions from the manufacturer and then were analysed using the Ceveron alpha Coagulation Analyser (Technoclone).

Procoagulant neutrophils and NDMPs

Thirty millilitre of the blood sample were transferred into a polypropylene tube that contained sodium citrate (3.8 g/dl, 1–9 vol blood) (Sigma). Peripheral blood neutrophils were separated by density-gradient centrifugation in a Polymorphprep (Nycomed) tube according to the manufacturer's instructions [20,21]. Briefly, the whole blood was carefully layered over one volume of Polymorphprep in a polypropylene tube. After centrifugation at 450 g for 35 min at 20°C, the neutrophil fraction was harvested using a pipette and the separated cells were then washed in RPMI-1640 medium (Sigma) by re-centrifugation at 600 g for 10 min at 20°C. Neutrophil purity, which was determined by flow cytometry using an anti-CD16 monoclonal antibody conjugated with FITC (eBioscience) and according to the cell distribution in forward and sideward scatter, was 95%. The cell free plasma was obtained by centrifugation at 10000 g for 30 min at 4°C. Isolated neutrophils were resuspended in the cell free plasma [i.e., neutrophil-rich plasma (NRP)], adjusted to 5×106 cells/ml and then maintained at 4°C for no more than 1 h before use.

In some experiments, various stimuli, such as 100 μg/ml lipopolysaccharide (LPS, a principal component of the outer membrane of Gram-negative bacteria) (Sigma) and 1 μM PMA (a protein kinase C activator) (Sigma) were added to the NRP, which was then warmed to 37°C for 2 h. After treatment with LPS or PMA, NDMP-rich supernatant and neutrophil pellets were separated by centrifugation at 400 g for 10 min. NDMPs were separated from the supernatant by filtrating using a 0.2 μm filtration device, Ceveron MFU-500 (Technoclone), and then resuspended in the cell free plasma. Additionally, neutrophil pellets were resuspended and adjusted to 5×106/ml in the cell-free plasma.

Plasma containing NDMPs or neutrophils was incubated with a saturating concentration of cyanine 5 (Cy5)-conjugated annexin V (BioVision) and phycoerythrin (PE)-conjugated monoclonal anti-human TF (CD142) antibody (American Diagnostica), in darkness for 30 min at 37°C. Neurtophils and NDMPs are significantly different in size and thus could be identified by their distinct distribution in the forward compared with sideward scatter dot plots. The procoagulant NDMPs were described as TF+ and/or annexin V+/CD16+ particles exhibiting forward light scatter equivalent to less than 1 μm (G1 gate), as defined by fluorescent standardized beads (0.5, 0.9 and 3.0 μm) (Megamix, Biocytex) (Figure 1) [22,23]. For microparticle quantification, we used a modification of the methods of Michelson et al. [24] in which CD16-positive particles were counted first and acquisition was stopped after 10000 events were counted under ‘high-run condition’ (0.06 ml/min). The total NDMP number (N in NDMP per ml) was calculated using the following formula: N=C × 104 × 10/[T × (0.06/min)], where ‘C’ represents the percentage NDMP (the number of NDMP as a percentage of the total number of CD16-positive particles), ‘T’ represents the total acquisition time, and 10 is a dilution factor.

Flow cytometric analysis of AIT effect on main procoagulant biomarkers on neutrophil (Neu) and NDMP in untreated (basal) (A) and LPS- and PMA-treated (B–E) NRP

Figure 1
Flow cytometric analysis of AIT effect on main procoagulant biomarkers on neutrophil (Neu) and NDMP in untreated (basal) (A) and LPS- and PMA-treated (B–E) NRP

Neutophils and NDMPs are significantly different in size and thus could be identified by their distinct distribution in the forward (FSC) compared with sideward (SSC) scatter dot plots. NDMPs exhibiting forward light scatter equivalent to less than 1 μm, as defined by standardized beads (0.5, 0.9 and 3.0 μm) (A). Annexin V–Cy5, cyanine 5-conjugated annexin V; Post-HE, hypoxic exercise test after AIT; Post-R, resting after AIT; Pre-HE, hypoxic exercise test before AIT; Pre-R, resting before AIT; TF–PE, phycoerythrin-conjugated monoclonal anti-human TF antibody.

Figure 1
Flow cytometric analysis of AIT effect on main procoagulant biomarkers on neutrophil (Neu) and NDMP in untreated (basal) (A) and LPS- and PMA-treated (B–E) NRP

Neutophils and NDMPs are significantly different in size and thus could be identified by their distinct distribution in the forward (FSC) compared with sideward (SSC) scatter dot plots. NDMPs exhibiting forward light scatter equivalent to less than 1 μm, as defined by standardized beads (0.5, 0.9 and 3.0 μm) (A). Annexin V–Cy5, cyanine 5-conjugated annexin V; Post-HE, hypoxic exercise test after AIT; Post-R, resting after AIT; Pre-HE, hypoxic exercise test before AIT; Pre-R, resting before AIT; TF–PE, phycoerythrin-conjugated monoclonal anti-human TF antibody.

Dynamic TG assay

Dynamic TG was measured by calibrated, automatic thrombinography (Synapse Thrombinoscope), as described in our previous studies [2,22,23]. Briefly, 80 μl of various plasma samples were placed in the wells of round bottom 96-well microtitre plates (Nunc). Coagulation was started by adding 0.1 M CaCl2 (20 μl) in a fresh mixture of fluobuffer (containing 20 mM HEPES and 60 mg/ml BSA, pH 7.35) containing 2.5 mM X-G-G-R-AMC (7-amino-4-methylcoumarin; the fluorogenic substrate; Synapse/Thrombinoscope). Upon cleaving by thrombin, the fluorescent AMC is released and measured with a 390-nm-excitation and a 460-nm-emission filter set in an Ascent Fluoroskan (Thermo Fisher Scientific). All reagents were warmed to 37°C before the experiment began. Fluorescence was recorded for 90 min. The fluorescence signal was corrected for substrate consumption, plasma colour variability and inner filter fluorescence effect by running in parallel calibrating wells where 80 μl of plasma samples were mixed with 20 μl of thrombin calibrator from Thrombinoscope.

The following parameters were recorded: (i) endogenous thrombin potential (ETP): area under the curve, which stands for the total amount of thrombin generated over the time; (ii) TG lag time: time to burst of TG, which roughly represents the clotting time; (iii) TG peak height: the highest thrombin concentration reached during the time course of thrombin formation and inhibition; and (iv) TG rate: peak height divided by the difference between time to peak and lag time, which represents the initial slope of TG [2,22,23].

Statistical analysis

Results are expressed as means±S.E.M. The statistical software package StatView was used for data analysis. Kolmogorov-Smirnov's goodness-of-fit test was used and normal distribution was observed in the present study. Experimental results were analysed by three (groups) × four (time sample points) repeated measures ANOVA and Bonferonni's post-hoc test to compare the cell counts, coagulant factors, neutrophils and NDMPs characteristics and dynamic TG parameters before and immediately after HE at the beginning of the present study and after 5 weeks in various groups. In addition, the comparison of cardiopulmonary fitness during GXT at the beginning of the present study and 5 weeks later in various groups were analysed by 3 (groups) × 2 (time sample points) repeated measures ANOVA and Bonferonni's post-hoc test. The criterion for significance was P<0.05.

RESULTS

Cardiopulmonary fitness and haematological parameters

Anthropometric variables did not significantly differ among the three groups at the beginning of the study (Table 1). Both AIT and MCT for the 5 weeks increased their work-rate, exercise time, V̇E, V̇O2 and V̇CO2 at peak exercise performance (Table 1, P<0.05). However, CTL for 5 weeks did not influence these cardiopulmonary responses to GXT (Table 1).

Acute bout of 12% O2 exercise significantly increased erythrocyte, neutrophil, lymphocyte, monocyte and platelet counts in blood (P<0.05, Table 2). However, the counts of various blood cells before or after the HE test remained unchanged following the 5-week intervention with AIT, MCT or CTL (Table 2).

Table 2
Effects of various exercise regimens on blood cell counts

Values are means±S.E.M. *P<0.05, Rest compared with HE. Post, after the intervention; Pre, before the intervention.

AITMCTCTL
ParameterPrePostPrePostPrePost
Erythrocyte (106/μl) 
 Rest 5.12±0.12 4.93±0.09 5.10±0.11 5.04±0.10 5.18±0.10 5.09±0.12 
 HE 5.49±0.10* 5.29±0.11* 5.47±0.10* 5.32±0.10* 5.55±0.12* 5.34±0.12* 
Neutrophil (103/μl)       
 Rest 2.82±0.19 2.73±0.16 2.97±0.26 3.11±0.24 2.93±0.27 2.72±0.27 
 HE 3.77±0.25* 3.41±0.24* 3.85±0.33* 3.75±0.36* 3.96±0.35* 3.70±0.33* 
Lymphocyte (103/μl) 
 Rest 2.43±0.18 2.47±0.24 1.74±0.13 1.96±0.10 2.08±0.17 2.09±0.15 
 HE 3.14±0.24* 2.92±0.24* 3.28±0.28* 2.97±0.18* 3.24±0.23* 3.26±0.19* 
Monocyte (103/μl) 
 Rest 0.53±0.04 0.55±0.07 0.57±0.04 0.65±0.04 0.56±0.06 0.55±0.06 
 HE 0.77±0.08* 0.73±0.08* 0.82±0.08* 0.75±0.07* 0.81±0.09* 0.76±0.06* 
Platelet (103/μl) 
 Rest 233±16 227±14 252±20 245±13 254±18 244±14 
 HE 285±19* 272±15* 296±17* 284±17* 310±18* 295±18* 
AITMCTCTL
ParameterPrePostPrePostPrePost
Erythrocyte (106/μl) 
 Rest 5.12±0.12 4.93±0.09 5.10±0.11 5.04±0.10 5.18±0.10 5.09±0.12 
 HE 5.49±0.10* 5.29±0.11* 5.47±0.10* 5.32±0.10* 5.55±0.12* 5.34±0.12* 
Neutrophil (103/μl)       
 Rest 2.82±0.19 2.73±0.16 2.97±0.26 3.11±0.24 2.93±0.27 2.72±0.27 
 HE 3.77±0.25* 3.41±0.24* 3.85±0.33* 3.75±0.36* 3.96±0.35* 3.70±0.33* 
Lymphocyte (103/μl) 
 Rest 2.43±0.18 2.47±0.24 1.74±0.13 1.96±0.10 2.08±0.17 2.09±0.15 
 HE 3.14±0.24* 2.92±0.24* 3.28±0.28* 2.97±0.18* 3.24±0.23* 3.26±0.19* 
Monocyte (103/μl) 
 Rest 0.53±0.04 0.55±0.07 0.57±0.04 0.65±0.04 0.56±0.06 0.55±0.06 
 HE 0.77±0.08* 0.73±0.08* 0.82±0.08* 0.75±0.07* 0.81±0.09* 0.76±0.06* 
Platelet (103/μl) 
 Rest 233±16 227±14 252±20 245±13 254±18 244±14 
 HE 285±19* 272±15* 296±17* 284±17* 310±18* 295±18* 

Intrinsic and extrinsic coagulation systems

At the beginning of the study, acute HE shortened aPTT along with elevating FVIII and fibrinogen concentrations (Table 3, P<0.05) despite unchanging PT, FV, FVII and protein C levels in plasma. However, either AIT or MCT attenuated the extents of aPTT shortening and FVIII/fibrinogen elevations induced by HE (Table 3). Additionally, no significant changes in intrinsic/extrinsic clotting time and coagulant factor levels in plasma were observed following CTL for 5 weeks (Table 3).

Table 3
Effects of various exercise regimens on blood coagulation system

Values are means±S.E.M. *P<0.05, Rest compared with HE, +P<0.05, Pre compared with Post. Post, after the intervention; Pre, before the intervention.

AITMCTCTL
ParameterPrePostPrePostPrePost
PT (s) 
 Rest 15.5±0.4 15.8±0.5 15.2±0.3 14.6±0.4 15.4±0.3 15.2±0.5 
 HE 14.6±0.6 15.7±0.7 14.7±0.5 15.4±0.5 15.1±0.7 15.0±0.8 
aPTT (s) 
 Rest 33.9±0.6 34.8±0.7 34.2±0.7 35.5±0.9 33.5±0.6 34.2±0.7 
 HE 29.1±0.5* 32.7±0.6*+ 29.9±0.6 32.4±0.2*+ 29.3±1.0* 30.3±1.1* 
Factor V (%) 
 Rest 102.1±5.1 95.3±5.1 99.2±5.9 98.4±4.1 102.2±5.2 105.7±6.3 
 HE 103.2±6.2 96.4±4.2 97.1±5.2 96.1±4.9 104.0±4.7 108.0±6.5 
Factor VII (%) 
 Rest 98.9±6.5 97.9±7.1 99.2±8.1 97.3±7.1 98.4±8.9 101.1±9.7 
 HE 100.4±5.8 99.1±7.3 101.4±9.1 102.3±6.3 101.5±9.0 103.2±9.5 
Factor VIII (%) 
 Rest 99.8±8.2 97.3±9.1 102.1±7.8 104.2±8.2 99.5±9.1 97.4±8.6 
 HE 167.2±16.1* 126.2±12.8*+ 185.3±12.7* 141.1±12.8*+ 187.4±15.3* 178.6±17.1* 
Protein C (%) 
 Rest 92.8±5.3 92.4±6.1 96.2±6.8 94.4±6.7 97.1±6.5 93.1±6.4 
 HE 96.7±6.1 98.6±7.1 95.3±7.6 96.6±9.1 99.4±6.2 97.5±6.8 
Fibrinogen (g/l) 
 Rest 2.53±0.32 2.76±0.31 2.62±0.29 2.97±0.31 2.52±0.21 2.74±0.19 
 HE 3.34±0.25* 2.81±0.21+ 3.38±0.32* 3.15±0.33 3.17±0.31* 3.24±0.21* 
AITMCTCTL
ParameterPrePostPrePostPrePost
PT (s) 
 Rest 15.5±0.4 15.8±0.5 15.2±0.3 14.6±0.4 15.4±0.3 15.2±0.5 
 HE 14.6±0.6 15.7±0.7 14.7±0.5 15.4±0.5 15.1±0.7 15.0±0.8 
aPTT (s) 
 Rest 33.9±0.6 34.8±0.7 34.2±0.7 35.5±0.9 33.5±0.6 34.2±0.7 
 HE 29.1±0.5* 32.7±0.6*+ 29.9±0.6 32.4±0.2*+ 29.3±1.0* 30.3±1.1* 
Factor V (%) 
 Rest 102.1±5.1 95.3±5.1 99.2±5.9 98.4±4.1 102.2±5.2 105.7±6.3 
 HE 103.2±6.2 96.4±4.2 97.1±5.2 96.1±4.9 104.0±4.7 108.0±6.5 
Factor VII (%) 
 Rest 98.9±6.5 97.9±7.1 99.2±8.1 97.3±7.1 98.4±8.9 101.1±9.7 
 HE 100.4±5.8 99.1±7.3 101.4±9.1 102.3±6.3 101.5±9.0 103.2±9.5 
Factor VIII (%) 
 Rest 99.8±8.2 97.3±9.1 102.1±7.8 104.2±8.2 99.5±9.1 97.4±8.6 
 HE 167.2±16.1* 126.2±12.8*+ 185.3±12.7* 141.1±12.8*+ 187.4±15.3* 178.6±17.1* 
Protein C (%) 
 Rest 92.8±5.3 92.4±6.1 96.2±6.8 94.4±6.7 97.1±6.5 93.1±6.4 
 HE 96.7±6.1 98.6±7.1 95.3±7.6 96.6±9.1 99.4±6.2 97.5±6.8 
Fibrinogen (g/l) 
 Rest 2.53±0.32 2.76±0.31 2.62±0.29 2.97±0.31 2.52±0.21 2.74±0.19 
 HE 3.34±0.25* 2.81±0.21+ 3.38±0.32* 3.15±0.33 3.17±0.31* 3.24±0.21* 

Procoagulant neutrophils and NDMPs

The flow cytometric analysis of various exercise regimen effects on main procoagulant biomarkers [i.e., TF expression and annexin V staining (PS exposure)] on neutrophil and NDMP in untreated and LPS-/PMA-treated NRP are shown in Figures 2(A)–2(E). Treatment of PMA significantly increased total NDMP count (Figures 2A–2C, P<0.05) and elevated the percentages of TF-rich/PS-exposed NDMPs (Figures 3A–3I, P<0.05) and neutrophils (Figures 4A–4I, P<0.05) in NRP. Despite modestly increased percentages of TF-rich/PS-exposed NDMPs (Figures 3A–3I, P<0.05), treating NRP with LPS left the total NDMP count unchanged (Figures 2A–2C), as well as TF expression and PS exposure on neutrophils (Figures 4A–4I).

Comparison of the effects of various exercise regimens on total NDMP counts in untreated (basal) and LPS- and PMA-treated NRP

Figure 2
Comparison of the effects of various exercise regimens on total NDMP counts in untreated (basal) and LPS- and PMA-treated NRP

(A) AIT group; (B) MCT group; (C) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Figure 2
Comparison of the effects of various exercise regimens on total NDMP counts in untreated (basal) and LPS- and PMA-treated NRP

(A) AIT group; (B) MCT group; (C) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Comparison of the effects of various exercise regimens on main procoagulant biomarkers (i.e., TF expression and PS exposure) of NDMPs in untreated (basal) and LPS- and PMA-treated NRP

Figure 3
Comparison of the effects of various exercise regimens on main procoagulant biomarkers (i.e., TF expression and PS exposure) of NDMPs in untreated (basal) and LPS- and PMA-treated NRP

(A, D and G) AIT group; (B, E and H) MCT group; (C, F and I) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Figure 3
Comparison of the effects of various exercise regimens on main procoagulant biomarkers (i.e., TF expression and PS exposure) of NDMPs in untreated (basal) and LPS- and PMA-treated NRP

(A, D and G) AIT group; (B, E and H) MCT group; (C, F and I) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. +P<0.05, Pre-R or Pre-HE compared with Post-R or Post-HE.

Comparison of the effects of various exercise regimens on main procoagulant biomarkers (i.e., TF expression and PS exposure) of neutrophils in untreated (basal) and LPS- and PMA-treated NRP

Figure 4
Comparison of the effects of various exercise regimens on main procoagulant biomarkers (i.e., TF expression and PS exposure) of neutrophils in untreated (basal) and LPS- and PMA-treated NRP

(A, D and G) AIT group; (B, E and H) MCT group; (C, F and I) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Figure 4
Comparison of the effects of various exercise regimens on main procoagulant biomarkers (i.e., TF expression and PS exposure) of neutrophils in untreated (basal) and LPS- and PMA-treated NRP

(A, D and G) AIT group; (B, E and H) MCT group; (C, F and I) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

At the beginning of the study, acute 12% O2 exercise markedly enhanced total NDMP counts in untreated and LPS-/PMA-treated NRP (Figures 2A–2C, P<0.05). Furthermore, the HE test also increased the percentages of TF expression without (Figures 4A–4C, P<0.05) or with (Figures 4G–4I, P<0.05) PS exposure on PMA-treated neutrophils.

Five weeks of AIT and MCT reduced the extents of the HE-promoted release of total microparticles from neutrophils under untreated and LPS-/PMA-treated conditions (Figures 2A–2C, P<0.05). Simultaneously, the two exercise regimens also decreased the percentages of TF expression without or with PS exposure on NDMPs (Figures 3A, 3G and 3H, P<0.05) and neutrophils (Figures 4A, 4B, 4G and 4H, P<0.05) in LPS- and PMA-treated NRP at rest status and following HE test. However, no significant changes in total NDMP count and TF expression/PS exposure on NDMPs and neutrophils in untreated and LPS-/PMA-treated NRP were observed following CTL for 5 weeks (Figures 24).

Dynamic TG in NPP and NRP

Figures 5 and 6 show the samples of HE-mediated dynamic TG in untreated and LPS-/PMA-treated NRP following 5 weeks of AIT respectively. With respect to the analytical parameters of dynamic TG, acute HE increased the ETP, peak height and rate of TG (P<0.05) but did not change the lag time of TG in plasma or NRP before the intervention (Table 4).

The calibrated, automatic thrombinographic analysis of AIT effect on dynamic TG in the cell-free plasma (plasma) or NRP

Figure 5
The calibrated, automatic thrombinographic analysis of AIT effect on dynamic TG in the cell-free plasma (plasma) or NRP

Post-HE, hypoxic exercise test after AIT; Post-R, resting after AIT; Pre-HE, hypoxic exercise test before AIT; Pre-R, resting before AIT.

Figure 5
The calibrated, automatic thrombinographic analysis of AIT effect on dynamic TG in the cell-free plasma (plasma) or NRP

Post-HE, hypoxic exercise test after AIT; Post-R, resting after AIT; Pre-HE, hypoxic exercise test before AIT; Pre-R, resting before AIT.

The calibrated, automatic thrombinographic analysis of AIT effect on dynamic TG in LPS- PMA-treated plasma that contained neutrophils (Neu), NDMP or Neu plus NDMP (Neu/NDMP)

Figure 6
The calibrated, automatic thrombinographic analysis of AIT effect on dynamic TG in LPS- PMA-treated plasma that contained neutrophils (Neu), NDMP or Neu plus NDMP (Neu/NDMP)

Post-HE, hypoxic exercise test after AIT; Post-R, resting after AIT; Pre-HE, hypoxic exercise test before AIT; Pre-R, resting before AIT.

Figure 6
The calibrated, automatic thrombinographic analysis of AIT effect on dynamic TG in LPS- PMA-treated plasma that contained neutrophils (Neu), NDMP or Neu plus NDMP (Neu/NDMP)

Post-HE, hypoxic exercise test after AIT; Post-R, resting after AIT; Pre-HE, hypoxic exercise test before AIT; Pre-R, resting before AIT.

Table 4
Effects of various exercise regimens on dynamic TG in plasma and NRP

Values are means±S.E.M. *P<0.05, Rest compared with HE, +P<0.05, Pre compared with Post. Post, after the intervention; Pre, before the intervention.

AITMCTCTL
ParameterPrePostPrePostPrePost
Plasma 
 Lag time (min)       
  Rest 9.0±1.2 9.4±0.6 7.9±0.4 8.6±0.7 9.1±1.1 9.2±0.9 
  HE 8.0±0.9 9.1±0.4 7.5±0.5 8.0±0.6 8.5±0.8 9.4±0.8 
 ETP (mm × min)       
  Rest 833±173 628±84 778±143 665±86 823±173 759±104 
  HE 1320±146* 891±114*+ 1146±125* 866±114*+ 1345±129* 1247±130* 
 Peak height (mm)       
  Rest 30.5±6.3 25.2±4.9 28.6±5.2 26.2±3.0 29.9±5.9 30.3±4.2 
  HE 53.4±6.8* 31.3±5.3*+ 51.6±7.1* 31.3±5.2*+ 53.8±6.2* 51.1±8.7* 
 Rate (mm/min)       
  Rest 1.8±0.4 1.3±0.3 2.1±0.5 1.6±0.3 2.1±0.5 2.4±0.3 
  HE 4.0±0.6* 2.6±0.3*+ 4.5±0.9* 2.7±0.6*+ 4.7±0.7* 4.7±0.7* 
NRP 
 Lag time (min)       
  Rest 6.6±0.2 6.9±0.2 5.6±0.4 6.0±0.5 6.0±0.5 6.2±0.3 
  HE 6.1±0.4 6.2±0.4 5.5±0.3 5.5±0.4 5.9±0.4 6.0±0.4 
 ETP (mM x min)       
  Rest 1262±134 1099±120 1164±102 1024±67 1279±94 1251±109 
  HE 1630±114* 1241±166+ 1455±92* 1155±88+ 1589±93* 1497±104* 
 Peak height (mm)       
  Rest 101.7±10.9 61.5±14.2+ 100.2±12.8 61.5±10.8+ 104.2±13.0 100.4±10.8 
  HE 136.5±10.6* 77.8±17.2+ 137.5±10.8* 77.8±11.8+ 140.1±9.1* 131.5±12.2* 
 Rate (mm/min)       
  Rest 10.7±1.5 6.9±1.2+ 14.1±3.3 7.5±1.6+ 14.5±3.3 11.9±1.5 
  HE 19.2±2.0* 8.0±1.7+ 20.2±3.1* 8.0±2.8+ 20.9±1.0* 18.6±2.0* 
AITMCTCTL
ParameterPrePostPrePostPrePost
Plasma 
 Lag time (min)       
  Rest 9.0±1.2 9.4±0.6 7.9±0.4 8.6±0.7 9.1±1.1 9.2±0.9 
  HE 8.0±0.9 9.1±0.4 7.5±0.5 8.0±0.6 8.5±0.8 9.4±0.8 
 ETP (mm × min)       
  Rest 833±173 628±84 778±143 665±86 823±173 759±104 
  HE 1320±146* 891±114*+ 1146±125* 866±114*+ 1345±129* 1247±130* 
 Peak height (mm)       
  Rest 30.5±6.3 25.2±4.9 28.6±5.2 26.2±3.0 29.9±5.9 30.3±4.2 
  HE 53.4±6.8* 31.3±5.3*+ 51.6±7.1* 31.3±5.2*+ 53.8±6.2* 51.1±8.7* 
 Rate (mm/min)       
  Rest 1.8±0.4 1.3±0.3 2.1±0.5 1.6±0.3 2.1±0.5 2.4±0.3 
  HE 4.0±0.6* 2.6±0.3*+ 4.5±0.9* 2.7±0.6*+ 4.7±0.7* 4.7±0.7* 
NRP 
 Lag time (min)       
  Rest 6.6±0.2 6.9±0.2 5.6±0.4 6.0±0.5 6.0±0.5 6.2±0.3 
  HE 6.1±0.4 6.2±0.4 5.5±0.3 5.5±0.4 5.9±0.4 6.0±0.4 
 ETP (mM x min)       
  Rest 1262±134 1099±120 1164±102 1024±67 1279±94 1251±109 
  HE 1630±114* 1241±166+ 1455±92* 1155±88+ 1589±93* 1497±104* 
 Peak height (mm)       
  Rest 101.7±10.9 61.5±14.2+ 100.2±12.8 61.5±10.8+ 104.2±13.0 100.4±10.8 
  HE 136.5±10.6* 77.8±17.2+ 137.5±10.8* 77.8±11.8+ 140.1±9.1* 131.5±12.2* 
 Rate (mm/min)       
  Rest 10.7±1.5 6.9±1.2+ 14.1±3.3 7.5±1.6+ 14.5±3.3 11.9±1.5 
  HE 19.2±2.0* 8.0±1.7+ 20.2±3.1* 8.0±2.8+ 20.9±1.0* 18.6±2.0* 

Five weeks of AIT and MCT significantly decreased HE-mediated ETP and peak height and rate of TG in plasma (Table 4, P<0.05), as well as resting and HE-mediated dynamic TG parameters in NRP (Table 4, P<0.05). Furthermore, the two exercise regimens also markedly attenuated the extents of HE-enhanced ETP and peak height and rate of TG in LPS- (Figure 7, P<0.05) or PMA-(Figure 8, P<0.05) treated plasma that contained neutrophils, NDMPs or neutrophils plus NDMPs. However, CTL did not significantly change the values of resting and HE-mediated dynamic TG parameters in untreated and LPS-/PMA-treated NRP (Figures 7 and 8).

Comparison of the effects of various exercise regimens on dynamic TG in LPS-treated plasma that contained neutrophils (Neu), NDMP, or Neu plus NDMP (Neu/NDMP)

Figure 7
Comparison of the effects of various exercise regimens on dynamic TG in LPS-treated plasma that contained neutrophils (Neu), NDMP, or Neu plus NDMP (Neu/NDMP)

(A, D, G and J) AIT group; (B, E, H and K) MCT group; (C, F, I and L) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Figure 7
Comparison of the effects of various exercise regimens on dynamic TG in LPS-treated plasma that contained neutrophils (Neu), NDMP, or Neu plus NDMP (Neu/NDMP)

(A, D, G and J) AIT group; (B, E, H and K) MCT group; (C, F, I and L) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Comparison of the effects of various exercise regimens on dynamic TG in PMA-treated plasma that contained neutrophils (Neu), NDMP, or Neu plus NDMP (Neu/NDMP)

Figure 8
Comparison of the effects of various exercise regimens on dynamic TG in PMA-treated plasma that contained neutrophils (Neu), NDMP, or Neu plus NDMP (Neu/NDMP)

(A,D,G and J) AIT group; (B, E, H and K) MCT group; (C, F, I and L) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

Figure 8
Comparison of the effects of various exercise regimens on dynamic TG in PMA-treated plasma that contained neutrophils (Neu), NDMP, or Neu plus NDMP (Neu/NDMP)

(A,D,G and J) AIT group; (B, E, H and K) MCT group; (C, F, I and L) CTL group. Post-HE, hypoxic exercise test after the intervention; Post-R, resting after the intervention; Pre-HE, hypoxic exercise test before the intervention; Pre-R, resting before the intervention. Values are means±S.E.M. *P<0.05, Pre-R or Post-R compared with Pre-HE or Post-HE; +P<0.05, Pre-HE compared with Post-HE.

DISCUSSION

The present investigation clearly demonstrated that an acute bout of 12% O2 exercise (i) shortened aPTT associated with increased FVIII and fibrinogen concentrations in plasma and (ii) accelerated endogenous TG in NRP related to enhanced TF expression and PS exposure on NDMPs and neutrophils. Notably, the present study was the first to observe that both AIT and MCT for 5 weeks attenuated the extents of TF-rich/PS-exposed NDMP formation and neutrophil/NDMP-mediated dynamic TG promoted by HE, which were accompanied by reduced FVIII and fibrinogen concentrations and lengthened intrinsic coagulation time in plasma. Hence, two exercise regimens effectively ameliorate neutrophil/NDMP-promoted dynamic TG possibly by down-regulating expression of procoagulant factors during HE, which may reduce the risk of inflammatory thrombosis evoked by hypoxic stress.

Aerobic capacity

Under isovolumic exercise protocols designed in the present study, AIT for 5 weeks effectively improved the capacity for O2 transport and utilization during exercise, that reflected higher values of V̇E and V̇O2 at peak performance, compared with MCT. Recently, the authors’ studies using healthy sedentary males [17] or patients with HF [15,16] revealed that the AIT regimen enhanced central and peripheral haemodynamic responses to exercise, apparently by increasing heart pumping efficiency and the perfusion and O2 extraction of skeletal muscles or cerebral tissues. Wisløff et al. [25] further demonstrated that AIT improved mitochondrial biogenesis, as reflected by the increased peroxisome-proliferative-activated receptor-γ co-activator-1α level of skeletal muscle in HF patients. Accordingly, the superior ventilatory, haemodynamic and metabolic adaptations induced by AIT may significantly contribute to improved exercise performance in healthy or diseased individuals.

Coagulant factors, NDMPs and TG mediated by HE

Extremely hypoxic environments are associated with increased incidence of vascular thromboemoblic events [1]. This hypercoagulable state is related to acute mountain sickness and cardiopulmonary disorders [3,4]. Blood is subjected to oxidative stress during extremely hypoxic exposure [2]; elevated oxidative stress may contribute to the activation of neutrophils [20,21] and the coagulation system [2]. Acute exposure to 12% O2 was observed to promote the chemotactic, phagocytic and oxidative burst activities of neutrophils, possibly by increasing lipid peroxidation and decreasing antioxidative capacity [20]. Moreover, the hypoxic stress also increased plasma FVIII level/activity and accelerated endogenous TG, which was accompanied by increased urinary 15-F2t-8-isoprostane level and decreased plasma total antioxidant content and superoxide dismutase activity [2]. However, these hypoxic effects on innate immunity [20] and coagulation [2] were ameliorated by pre-treatment with antioxidant vitamin E. The present study further demonstrated that acute 12% O2 exercise (i) shortened aPTT with elevating FVIII concentration but did not influenced PT and FVII level and (ii) increased endogenous TG rate without changing lag time in plasma, suggesting the HE test accelerates blood clotting time by activating the intrinsic coagulation pathway.

Addition of neutrophils into plasma exhibited the shortened lag time and increased peak height and rate of TG noted in the present study. By simulating inflammatory conditions using pre-treatment with LPS and PMA, these stimuli were shown to increase the levels of TF-rich and/or PS-exposed NDMPs and neutrophils in NRP. Furthermore, acute HE markedly increased the release of total microparticles from stimulated neutrophils and the formation of procoagulant NDMPs and neutrophils in NRP, which were accompanied by increased ETP and elevated peak height and rate of TG in NRP. Accordingly, HE may accelerate endogenous TG in NRP via increasing levels of procoagulant NDMPs and neutrophils under inflammatory conditions.

Effects of exercise training on HE-mediated coagulant factors, NDMPs and TG

In this investigation, either an AIT or MCT regimen attenuated the extents of aPTT shortening and FVIII/fibrinogen elevations caused by HE. Simultaneously, the two exercise regimens also depressed HE-promoted dynamic TG in plasma. Our recent study has demonstrated that HE significantly increased plasma myeloperoxidase concentration, whereas both AIT and MCT regimens manifestly lowered the peroxide level at rest and following HE [17]. Since hypoxia promotes FVIII-dependent TG by enhancing lipid peroxidation [2], the two exercise regimens may reduce HE-promoted intrinsic coagulation activation through depressing circulatory oxidative stress.

In basal and LPS-/PMA-treated NRP, 5 weeks of AIT and MCT also significantly lowered TF-rich/PS-exposed NDMP levels and depressed neutrophil/NDMP-mediated dynamic TG following HE. Wang, et al. [14] demonstrated that MIE (60% V̇O2max) suppressed platelet-neutrophil interaction by increasing neutrophil antioxidative capacity. Moreover, MIE also decreased the expressions of Mac-1 on neutrophils and P-selectin on platelets under LPS treatment [14]. Notably, prior exercise intensity lowered to 40% V̇O2max [warm-up exercise (WUE)] attenuated the enhancements of platelets binding to neutrophils and production of neutrophil reactive oxygen species promoted by heavy-intensity exercise (HIE, 80% V̇O2max) as with the change of platelet-neutrophil interaction by MIE [14]. Taken together, these findings suggest that the exercise regimen that consists of sustained MIE or HIE preceded by WUE might protect individuals against risk of neutrophil-related inflammatory thrombosis caused by HE.

Study limitation

Our small size in each group (n=20) is a major limitation of the present study. However, the aerobic capacity, coagulant factors, NDMPs and dynamic TG results obtained from this investigation have high values of statistical power from 0.915 to 1.000. Additionally, the subjects used tended to be young and healthy and thus further clinical evidence was required to extrapolate the present results to patients with abnormal or diseased immune and haemostatic systems.

Conclusions

Although the two exercise protocols were isovolumic at the same exercise duration, AIT exhibits a larger cardiopulmonary adaptation than the MCT does. However, both AIT and MCT groups de-sensitize the extent of dynamic TG in NRP following HE. These changes are likely to be mediated by reducing the releases of TF-rich/PS-exposed microparticles from neutrophils and lowering the TF expression and PS exposure on neutrophils/NDMPs. Moreover, the two exercise regimens simultaneously attenuate HE-induced activation of the intrinsic coagulation pathway by decreasing plasma FVIII and fibrinogen levels. Therefore, AIT can be considered an effective exercise strategy that improves aerobic capacity and simultaneously increases the resistance to thrombotic risk provoked by hypoxia.

Abbreviations

     
  • AIT

    aerobic interval training

  •  
  • AMC

    7-amino-4-methylcoumarin

  •  
  • aPTT

    activated partial thromboplastin time

  •  
  • CTL

    control

  •  
  • Cy5

    cyanine 5

  •  
  • ETP

    endogenous thrombin potential

  •  
  • GXT

    graded exercise test

  •  
  • HE

    hypoxic exercise test

  •  
  • HF

    heart failure

  •  
  • HIE

    heavy intensity exercise

  •  
  • HR

    heart rate

  •  
  • LPS

    lipopolysaccharide

  •  
  • MCT

    moderate continuous training

  •  
  • MIE

    moderate-intensity exercise

  •  
  • NDMP

    neutrophil-derived microparticle

  •  
  • NRP

    neutrophil-rich plasma

  •  
  • PE

    phycoerythrin

  •  
  • PS

    phosphatidylserine

  •  
  • PT

    prothrombin time

  •  
  • TF

    tissue factor

  •  
  • TG

    thrombin generation

  •  
  • V̇CO2

    carbon dioxide production

  •  
  • V̇E

    minute ventilation

  •  
  • V̇O2

    oxygen consumption

  •  
  • V̇O2max

    maximal V̇O2

  •  
  • WUE

    warm-up exercise

AUTHOR CONTRIBUTION

Jong-Shyan Wang was involved in the conception and design of research. Yi-Ching Chen and Ching-Wen Ho performed experiments. Jong-Shyan Wang, Yi-Ching Chen and Ching-Wen Ho analysed data, interpreted results of experiments, prepared the Figures and drafted the paper. Jong-Shyan Wang edited and revised the paper. Jong-Shyan Wang, Yi-Ching Chen, Ching-Wen Ho and Hsing-Hua Tsai approved the final version of the paper.

We would like to thank the volunteers for their enthusiastic participation.

FUNDING

This work was supported by the National Science Council of Taiwan [grant number NSC 100-2314-B-182-004-MY3]; the Chang Gung Medical Research Program [grant numbers CMRPD1A0132 and CMRPD2C0161]; and the Healthy Aging Research Center, Chang Gung University [grant number EMRPD1A0841].

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