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Vascular Nitric Oxide–Superoxide Balance and Thrombus Formation after Acute Exercise

PRZYBOROWSKI, KAMIL1; PRONIEWSKI, BARTOSZ1; CZARNY, JOANNA1; SMEDA, MARTA1; SITEK, BARBARA1; ZAKRZEWSKA, AGNIESZKA1; ZOLADZ, JERZY A.2; CHLOPICKI, STEFAN1,3

Medicine & Science in Sports & Exercise: July 2018 - Volume 50 - Issue 7 - p 1405–1412
doi: 10.1249/MSS.0000000000001589
BASIC SCIENCES
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Introduction An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)–dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity.

Methods At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide (O2) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography–based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF.

Results An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF concentrations.

Conclusion An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.

1Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, POLAND;

2Department of Muscle Physiology, Chair of Physiology and Biochemistry, Faculty of Rehabilitation, University School of Physical Education, Krakow, POLAND; and

3Chair of Pharmacology, Jagiellonian University Medical College, Krakow, POLAND

Address for correspondence: Stefan Chlopicki, M.D., Ph.D., Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland; E-mail: stefan.chlopicki@jcet.eu.

Submitted for publication November 2017.

Accepted for publication February 2018.

It is widely accepted that regular exercise activity and exercise training have beneficial effects on cardiovascular health, and there is a strong and inverse relationship between physical fitness and mortality in humans (1). Physical exercise represents an effective strategy for the prevention and treatment of cardiovascular diseases (2). There are multiple mechanisms responsible for chronic exercise-related health benefits, including the reduction of cardiovascular risk factors (e.g., hypertension, obesity, and insulin resistance) or exercise-induced improvement in endothelial function (3). It was reported in animal and human studies that exercise training augmented endothelium-dependent vasodilation (4–7). In general, regular exercise not only increases shear stress-induced production and release of nitric oxide (NO) but also reduces NO inactivation by upregulation of antioxidant defense mechanisms, ultimately leading to increased NO bioavailability (8).

However, in some circumstances, exercise can become harmful to the cardiovascular system and detrimental versus beneficial effects of exercise depend on type, intensity, and training workload as well as physical fitness and health status of individuals (9). It has been shown that frequent strenuous physical activity and regular strenuous running were associated with increased incidence of coronary heart disease, cerebrovascular disease, venous thromboembolism, and all-cause mortality (10,11). Furthermore, vigorous exercise and long-distance running can trigger acute myocardial infarction and sudden death (12–14). It was demonstrated in humans that a single bout of acute exercise with high intensity resulted in temporary endothelial dysfunction, as evidenced by a decrease in flow-mediated dilatation (FMD) immediately after exercise (15,16). With regard to animals, there are only a few reports showing the effect of a single bout of acute exercise on postexercise endothelial function in rats, but their results are conflicting, showing increased or decreased postexercise endothelium-dependent vasodilation (17–19).

Exercise is associated with increased production of reactive oxygen species (ROS) (20), which are also involved in the mechanism of muscle fatigue (21); thus, postexercise endothelial dysfunction could be due to exercise-induced oxidative stress (22). Namely, NO, an important endothelial mediator controlling thrombosis (23), can be readily inactivated by the superoxide anion (O2) (24). Therefore, the postexercise disturbances in NO bioavailability caused by ROS generated in response to acute intense exercise may lead to the impairment of NO-dependent antiplatelet mechanisms and higher cardiovascular risk. Indeed, acute intense and endurance exercise have been related to platelet activation and prothrombotic phenotype (25–27).

The purpose of the present study was to determine alterations in the balance between NO and O2 production in mice aorta at different time points after exhaustive running. In parallel, postexercise thrombus formation was measured in a flow-chamber system (total thrombus-formation analysis system [T-TAS]) with collagen-coated microchips, for analysis of platelet-dependent thrombogenicity. Specifically, we tested the hypothesis that postexercise increase in ROS generation corresponds with a decrease in NO bioavailability, resulting in an increased thrombotic risk at recovery time after acute exhaustive exercise.

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MATERIALS AND METHODS

Animals

Male C57BL/6 mice (Mossakowski Medical Research Centre, Warsaw, Poland) at the age of 4–6 months were used in the experiments. The animals were housed five to six mice per cage in a temperature- and humidity-controlled room with a 12-h light/dark cycle and unlimited access to drinking water and standard rodent food. Mice from each cage were divided randomly into sedentary and postexercise experimental groups at 0, 2, and 4 h after completion of exercise. All animal procedures were compliant with the Guide for the Care and Use of Laboratory Animals published by the European Parliament, Directive 2010/63/EU, and were approved by the local Ethical Committee on Animal Experiments.

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Experimental protocol

Briefly, 2 days before the experiments, mice were acclimatized to a closed two-line treadmill equipped with a shock grid (Columbus Instruments, Columbus, OH). Subsequently, the mice were subjected to an incremental running test on the treadmill at 0° incline. The treadmill was started at 5 m·min−1, and the speed was increased by 4 m·min−1 every 10 min until the mice reached exhaustion, defined as being unable to continue running for at least 5 s despite electrical stimulation (current 0.34 mA, voltage 25 V, electrical stimulation frequency 3 Hz). Simultaneously, sedentary mice were exposed to the treadmill chamber with shock grid without running.

Sedentary and exercised mice at time points of 0, 2, and 4 h after completion of a single bout of exhaustive exercise were anesthetized with ketamine and xylazine (100 and 10 mg·kg−1 i.p., respectively). Blood samples were drawn from the right heart ventricle into a syringe with dalteparin (10 U·mL−1), and aorta tissue was removed. Whole blood was used for blood cell count and platelet-dependent thrombogenicity in T-TAS, whereas isolated aorta was taken for measurement of vascular NO and O2 production. Furthermore, blood was centrifuged (1000g, 10 min, 4°C) to obtain plasma samples. Plasma samples were deep frozen (−80°C) and stored until biochemical analysis.

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Production of NO in the isolated aorta

Before the experiments, Krebs–HEPES buffer (consisting of, in mM, NaCl, 99.0; KCl, 4.7; MgSO4·7 H2O, 1.2; KH2PO4, 1.0; CaCl2·H2O, 2.5; NaHCO3, 25.0; glucose, 5.6; and Na-HEPES, 20.0) was filtered through a 0.22 μm paper syringe filter and equilibrated to pH 7.4. The filtered buffer was then deoxygenized by bubbling argon gas for at least 30 min. Diethyldithiocarbamic acid sodium salt (DETC) (3.6 mg) and FeSO4·7H2O (2.25 mg) were separately dissolved under argon gas bubbling in two 10 mL volumes of ice-cold Krebs–HEPES buffer and were kept under gas flow on ice until used. For measurements of NO, electron paramagnetic resonance (EPR) spin trapping with DETC was used. One half (upper segment) of freshly harvested aortas were cleaned from adherent tissue, opened longitudinally, and transferred into wells of a 48-well plate, each filled with 100 μL Krebs–HEPES and preincubated for 30 min at 37°C. DETC and FeSO4·7H2O solutions were mixed 1:1 (v/v) to obtain 250 μL Fe(DETC)2 colloid per well (final concentration 285 μM) and immediately added to the aorta in parallel with calcium ionophore A23187 (final concentration 1 μM) to stimulate eNOS and subsequently incubated at 37°C for 90 min.

After aorta incubation, each aorta was removed from the buffer, drained on a piece of wipe for 5 s, and the wet mass was measured. Next the aorta was frozen, in liquid nitrogen, into the middle of a 400 μL column of Krebs–HEPES buffer and stored at −80°C until measured. The frozen column was pushed out directly into a finger Dewar (Noxygen, Germany) containing liquid nitrogen. EPR spectra were obtained using an X-band EPR spectrometer (EMX Plus, Bruker, Germany), equipped with a rectangular resonator cavity H102. Signals were quantified by measuring the total amplitude of the NO-Fe(DETC)2 after correction of baseline. All samples were normalized to weight of wet aorta. There were no differences in the weight of aortas between groups, and the intra-assay coefficient of weight variation reached approximately 12%.

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Generation of O2 in the isolated aorta

Oxidation of dihydroethidium (DHE) to 2-OH-E+ was used as an indicator of superoxide radical anion (O2) production in aortas. One half (lower segment) of freshly harvested aortas were carefully cut out and cleaned from any adherent tissues and then opened longitudinally and transferred into wells of a 48-well plate, each well being filled with 100 μL of 10 μM DHE in phosphate-buffered saline (without Ca/Mg ions) pH 7.4 and incubated for 45 min at 37°C under dark conditions. After incubation, each aorta was removed from the buffer, drained on a piece of wipe for 5 s and immediately frozen in liquid nitrogen in light safe tubes, and stored at −80°C until use. Aorta samples were homogenized in 250 μL of 0.1% Triton X-100 in ice-cold Dulbecco’s phosphate-buffered saline buffer and then centrifuged for 5 min at 1000g at 4°C to obtain supernatant. To extract the oxidation products of DHE, 200 μL of supernatant was then transferred to a fresh tube and mixed with 100 μL of 0.2 M HClO4 in methanol. The samples were incubated on ice for 2 h and subsequently centrifuged for 30 min at 20,000g at 4°C. A volume of 120 μL of the supernatant was then transferred to a fresh tube containing 120 μL of 1 M phosphate buffer (pH 2.6). This solution was centrifuged for an additional 15 min at 20,000g at 4°C. A volume of 200 μL of the resulting supernatant was transferred to high-performance liquid chromatography (HPLC) vials for analysis. Measurement of 2-OH-E+ was performed by HPLC technique with fluorescence detection using the Ultimate3000 Dionex system (Thermo, USA). Chromatographic separation was conducted on an analytical column Kinetex C18 (4.6 × 100 mm, 2.6 μm; Phenomenex, Torrance, CA) with the oven temperature set at 40°C. The mobile phase consisted of acetonitrile (A) and water (B) both with an addition of 0.1% trifluoroacetic acid with the following linear eluting steps: 0.0 min (A:B, 25/75 v/v), 0.5 min (A:B, 25/75 v/v), 8 min (A:B, 35/65 v/v), 9 min (A:B, 95/5 v/v), 11 min (A:B, 95/5 v/v), 12.0 min (A:B, 25/75 v/v), and 14.0 min (A:B, 25/75 v/v). The flow rate was set at 1 mL·min−1. A sample volume of 50 μL was injected onto the column. The level of O2 generated in samples was calculated from a standard curve and normalized to total protein concentration.

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Evaluation of thrombogenicity in microchip-based flow-chamber system (T-TAS)

To analyze platelet-dependent thrombogenicity under flow conditions, a microchip-based flow-chamber system was used (T-TAS; Fujimori Kogyo Co., Ltd., Tokyo, Japan). For monitoring platelet thrombus formation, heparinized blood samples were immediately perfused over a PL-microchip coated with collagen at flow rates 12 μL·min−1, which created an initial wall shear rate of 1000 s−1. Thrombus formation within the microchip caused flow disturbances that resulted in pressure increase. Flow pressure changes were monitored during blood perfusion. The obtained flow pressure pattern for each sample was used to analyze thrombus formation based on the following parameters: time to onset of thrombus formation (T10, time required to reach the pressure of 10 kPa; min), occlusion time (OT, time required to reach the pressure 60 kPa; min), and area under the flow pressure curve for 10 min after the start of the assay, defined as total platelet-dependent thrombogenicity (AUC10, less than 60 kPa).

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Biochemical assays

Blood cell count was determined by a hematology analyzer ABC Vet (Horiba, Germany). For the measurement of PGI2, the plasma concentration of its stable metabolite 6-keto-PGF1α was determined using a commercially available ELISA kit according to the manufacturer’s instructions with 10 times diluted plasma samples. Plasma nitrite (NO2) and nitrate (NO3) were determined using an ENO-20 NOX analyzer (Eicom Corp., Kyoto, Japan) as described previously (28).

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Statistical analysis

Data represent individual measurements and median values ± interquartile ranges, unless otherwise stated. All results were verified for normality using Shapiro–Wilk’s test and variance homogeneity using Brown Forsythe’s and Levene’s tests. Data showing normal distribution and homoscedasticity of variance, including NO and O2 production, OT, plasma concentration of 6-keto-PGF, hemoglobin, and hematocrit, were analyzed for statistical significance by parametric one-way ANOVA followed by post hoc LSD Fischer’s or Bonferroni’s multiple comparisons tests. In turn, data showing departures from normal distributions and/or variance heteroscedasticity, including T10, area under the flow pressure curves (AUC), plasma concentration of nitrite and nitrate, and number of white blood cells, red blood cells, and platelets, were transformed to obtain normal distribution using the Box–Cox transformation, and the transformed values were further analyzed. All transformed results were verified for normality using Shapiro–Wilk’s test and variance homogeneity using Brown Forsythe’s and Levene’s tests. The significant differences in transformed data of nitrite and nitrate showing normal distribution and homoscedasticity of variance were tested by parametric one-way ANOVA followed by post hoc LSD Fischer’s multiple comparisons test. The transformed data of T10, AUC, white blood cells, red blood cells, and platelets still deviating from normal distribution and/or variance homogeneity were analyzed using the Kruskal–Wallis nonparametric test. Statistical analysis was performed in Statistica version 12.5. A P value <0.05 was considered statistically significant.

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RESULTS

Effect of acute exercise on postexercise NO and superoxide anion (O2) production in aorta

An acute bout of exhaustive running substantially decreased NO production in aorta measured 2 h after exercise as compared with sedentary mice (P < 0.01, Fig. 1A). However, 4 h after exercise, NO production returned to baseline level (P < 0.001 vs 2 h, Fig. 1A); thus, postexercise impairment of NO production was transient.

FIGURE 1

FIGURE 1

In turn, postexercise production of O2 in aorta was significantly increased by an acute bout of exhaustive running with a pronounced, significant rise 2 h after completion of exercise as compared with sedentary mice (P < 0.05, Fig. 1B). This increase in O2 production was followed by normalization to sedentary level 4 h after exercise, presenting a similar time-dependent pattern of changes as observed in the postexercise changes in the production of NO (Fig. 1A).

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Effect of acute exercise on postexercise platelet-dependent thrombogenicity

An acute bout of exhaustive running did not affect platelet activity as monitored by platelet thrombus formation in whole blood ex vivo. Parameters of platelet-dependent thrombogenicity analyzed in a microchip-based flow-chamber system including the time to start of thrombus formation in microchip (T10), OT, and AUC remained unchanged at the time points of 0, 2, and 4 h after completion of exercise as compared with sedentary mice (Fig. 2). As shown in Table 1, an acute bout of exhaustive running also had no influence on platelet count.

TABLE 1

TABLE 1

FIGURE 2

FIGURE 2

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Postexercise plasma concentrations of 6-keto-PGF, nitrite and nitrate

There were no significant changes in postexercise concentration of 6-keto-PGF and nitrate over time after completion of an acute bout of exhaustive running (Fig. 3A and C). However, the plasma concentration of nitrite was elevated immediately after completion of exercise (P < 0.05, Fig. 3B) and returned to baseline level at a time point of 2 h after exercise (Fig. 3B).

FIGURE 3

FIGURE 3

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DISCUSSION

In the present work, we demonstrated, to our knowledge, for the first time that in healthy mice the balance between production of NO and superoxide anions (O2) in the aorta is significantly altered 2 h after completion of a single bout of strenuous exercise, but it did not result in a postexercise platelet-dependent prothrombotic state. These results suggest that transient reduction in NO production, most likely linked to exercise-induced oxidative stress, representing an important aspect of vascular adaptation to intense exercise, does not compromise endothelial thromboresistance in healthy mice.

Our studies demonstrated that, in mice similarly as in humans, a single bout of strenuous exercise with high intensity caused transient endothelial dysfunction. In humans, this phenomenon was evidenced by a decrease in FMD observed immediately after high-intensity exercise, but not after moderate intensity exercise (15,16). In the present study performed in mice, we showed that a single bout of exhaustive running profoundly decreased NO production in aorta at the time point of 2 h after completion of exercise, and then returned to the basal level within 4 h postexercise. These results support the notion that exercise-induced postexercise endothelial response displays a transient pattern of changes, which have already been reported in humans. Literature data reviewed by Dawson et al. (9) highlighted that human endothelial function measured by FMD is impaired immediately after an acute bout of exercise, and this is followed by normalization at later time points. Our results in mice also emphasize that postexercise vascular effects depend on recovery time after exercise. The transient pattern of postexercise changes in NO production in mice aorta reported by us is in agreement with the study by Haram et al. (18), where the authors showed that acetylcholine-induced dilation of rat aorta was reduced immediately after exercise and then returned to baseline level within 6 h (18). Only a few studies have illustrated the effects of a single bout of acute exercise on endothelial function in rats (17–19). However, the data were conflicting, showing that a single bout of intense swimming and running caused a decrease or increase in acetylcholine-induced endothelium-dependent vasorelaxation immediately after exercise (17–19).

In the present work, we showed that the transient impairment of NO-dependent vascular function is linked to the overproduction of O2, which is compatible with the notion that physical exercise triggers generation of ROS and can lead to oxidative stress (29). The magnitude of exercise-induced oxidative stress that is also considered to be important for the induction of the beneficial effects of exercise (30) depends on several factors, including the type of exercise and its intensity (29). Oxidative stress generated directly in vessels can be considered an important trigger of postexercise NO-dependent endothelial dysfunction. This is supported by studies in rats, showing that a transient decrease in vascular dilation in response to acetylcholine demonstrated immediately after acute running was improved by incubating of aorta with O2 scavenger superoxide dismutase (18). Furthermore, the reduced maximal relaxation of aorta in response to acetylcholine immediately after high-intense swimming was simultaneously accompanied by a substantial increase in lipid peroxidation in aorta (19).

Our study extended the research of Haram et al. (18) and Brito et al. (19) and evaluated the time course of postexercise NO production in comparison with O2 production in mice aorta after an exhaustive running by direct measurements of NO and O2 using well-chosen EPR and DHE/HPLC-based methods, respectively. We showed that exercise induced a significant O2 overproduction in aorta 2 h after exercise, and this effect clearly corresponded to the impairment of NO production. Our results provide strong evidence that exercise-induced oxidative stress generated in aorta is responsible for postexercise endothelial dysfunction characterized by diminished NO bioavailability. Indeed, generated O2 directly inactivates NO by forming peroxynitrite, which in turn leads to decrease NO production (24). Enzymatic sources of postexercise ROS generation were not determined here and could involve NOX, uncoupled NO synthase, or other sources (31).

The important finding of this work was to show that in healthy mice, a postexercise decrease in NO bioavailability associated with increased O2 generation did not result in platelet activation and prothrombotic state. In previous studies, it was reported that an acute high-intensity exercise induced platelet hyperactivity, increasing the thrombotic risk (25,26). Similarly, significant platelet activation was found in athletes after marathon run and triathlon competitions (27). Given that endothelium-derived NO is a potent inhibitor of platelet function (23), we expected that postexercise decrease in NO bioavailability 2 h after exercise would result in increased platelet activation and prothrombotic state. Using a state-of-the art microchip-based flow-chamber system (T-TAS), which was reported to be a suitable technique for monitoring whole-blood thrombogenicity (32) and selectively thrombogenicity of platelets (33), we showed that an exhaustive running in mice did not affect collagen-induced platelet thrombus formation at any time points after completion of exercise, as evidenced by the time to onset of thrombus formation, capillary OT, and total platelet-dependent thrombogenicity. Given the observations that exercise can transiently increase platelet activity in humans (25–27), our results suggest that in mice, other mechanisms may compensate deficiency of endothelium-derived NO and protect against postexercise thrombosis. In our previous studies, we demonstrated overactivation of platelets in ApoE/LDLR−/− mice with atherosclerosis as compared with healthy mice. However, strenuous exercise did not further increase the platelet activation in ApoE/LDLR−/− mice but attenuated platelet activation that could be linked to upregulated prostacyclin (PGI2)-dependent antiplatelet mechanisms (28). It has been previously reported that exercise increases production of PGI2 (34) and the exercise-induced release of PGI2 significantly correlates with exercise capacity in humans (35). Furthermore, it has been postulated (35) that the exercise-induced increase in PGI2 might play an important role in decreasing the cardiovascular hazard of vigorous exercise. In our present study, we did not observe postexercise changes in PGI2 production as evidenced by unaltered plasma concentration of 6-keto-PGF, a stable metabolite of PGI2. However, this does not exclude the role of PGI2, which can safeguard thromboresistance in the settings of NO-deficiency (36).

The concentrations of nitrite and nitrate are commonly used as a marker of NO bioavailability and endothelial function (28,37). However, it has been recently reported by Majerczak et al. (38) that even a strenuous physical exercise in young healthy humans did not significantly change plasma concentrations of nitrite and nitrate, and the sum of plasma nitrite and nitrate concentrations, as compared with their levels at rest. In the present study, we demonstrated that plasma concentration of nitrite increased immediately after exercise and returned to basal level 2 h after exercise, whereas the postexercise plasma concentration of nitrate remained unchanged. This finding suggests that the increased plasma concentration of nitrite does not reflect NO production by aorta, but rather it could be a result of the activation of the nitrate–nitrite–NO reductive pathway, which can account for an alternative source of NO (39). It was reported in rats by Piknova et al. (40) that skeletal muscles represent a large reservoir for nitrate, where they are substantially reduced to nitrite during an acute exercise. This leads to speculation that the increase in nitrite after exercise observed in our study might be a result of nitrite release into blood from muscles. Furthermore, these data may suggest that nitrite produced during exercise in muscles can be important in maintenance of postexercise balance in NO bioavailability in circulation and in support of thromboresistance, especially in the setting of a transient impairment of NO-dependent endothelial function after an acute bout of exercise.

In conclusion, an acute bout of strenuous exercise reduced NO and increased O2 production in aorta. This response was most pronounced 2 h after completion of exercise. Surprisingly, the reduced NO and increased O2 production did not result in a postexercise platelet-dependent prothrombotic state. These results show that transient reduction in NO production alone, most likely linked to exercise-induced oxidative stress, does not provoke postexercise platelet hyperactivity in healthy mice. Apparently, in healthy mice, vascular adaptive mechanisms induced by intense exercise afford thromboresistance despite impairment in NO-dependent vascular function. Both NO produced in the nitrate–nitrite–NO reductive pathway as well as fully preserved PGI2 function could contribute to the protection against postexercise thrombotic events in the settings of transiently impaired NO-dependent function. Further studies to evidence mechanisms underlying the prevention of increased postexercise cardiovascular hazard due to maladaptive impairment of NO-dependent function after an acute exercise are warranted.

This work was supported by the National Science Centre, Poland, grant Preludium no. 2017/25/N/NZ4/02073.

The authors declare no conflicts of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine. The authors declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.

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Keywords:

THROMBOGENICITY; PROSTACYCLIN; ENDOTHELIUM; FLOW-CHAMBER SYSTEM

© 2018 American College of Sports Medicine