Effects of Short-term Exercise on Female Platelet Function During Different Phases of the Menstrual Cycle
Abstract Previous studies have shown that premenopausal women have a low incidence of cardiovascular diseases, and that acute exercise affects male platelet function in an intensity-dependent manner. To investigate whether acute exercise affects female platelet function differently from males, sixteen sedentary women in the midfollicular phase or midluteal phase received strenuous or moderate exercise on a bicycle ergometer. Before and immediately after exercise, platelet adhesiveness, adenosine diphosphate-induced platelet aggregation and intracellular calcium concentration elevation, platelet cAMP and cGMP contents, urinary 11-dehydro-TXB2 and 6-keto-prostaglandin F1α levels, and plasma nitric oxide metabolite level were determined. Our results showed no differences in exercise performance and in resting platelet function between two menstrual phases, with little change in urinary eicosanoid metabolites and platelet cAMP levels under all experimental conditions. In addition, for women in the midfollicular phase, (1) strenuous exercise increased platelet adhesiveness, adenosine-diphosphate-induced platelet aggregation, and intracellular calcium concentration elevation, whereas moderate exercise suppressed them; (2) moderate exercise enhanced plasma nitric oxide metabolite and platelet cGMP levels. In contrast, none of these platelet functions was affected by acute exercise in the midluteal phase. Therefore, we conclude that acute exercise affects female platelet function in an intensity-dependent manner in the midfollicular phase but not in the midluteal phase. The irresponsiveness of platelets to acute exercise in the luteal phase may partially explain why premenopausal women have a lower incidence of cardiovascular diseases than men.
- Received November 19, 1996.
- Accepted March 12, 1997.
Platelets play an important role in the pathogenesis and progression of cardiovascular diseases.1 2 In addition, vigorous short-term exercise may enhance the risk of major vascular thrombotic events and transiently increase the incidence of primary cardiac arrest.3 4 It is also known that premenopausal women have a lower incidence of cardiovascular diseases than men.5 Our previous study using healthy men and male patients with stable angina has shown that various intensities of short-term exercise affect blood platelet function differently, ie, moderate exercise desensitizes platelets whereas strenuous exercise potentiates them.6 How short-term exercise affects platelet function in females is totally unknown at present. We therefore conducted this study to clarify the effects of various intensities of exercise on platelet function in females during different phases of the menstrual cycle. Both strenuous exercise and moderate exercise were undertaken by sedentary healthy females who were in their midfollicular phase or midluteal phase. Platelet functional activity was determined by its adhesiveness and aggregability.
Although animal studies or studies using male subjects indicated that short-term exercise could enhance PGI2 and NO releases,7 8 9 similar studies using female subjects have not been reported. Both PGI2 and NO are potent antiplatelet agents with cAMP mediating the former effect and cGMP mediating the latter effect.10 11 These platelet cyclic nucleotides can reduce the agonist-induced rise of platelet [Ca2+]i and hence suppress agonist-induced platelet activation.12 In this study, we also determined plasma NO metabolite level, urinary PGI2 metabolite level, platelet cGMP and cAMP contents, and platelet [Ca2+]i to elucidate the possible underlying mechanism responsible for exercise-induced platelet functional changes.
The protocol had previously been reviewed and approved by an institutional committee for the protection of human subjects. Sixteen sedentary, healthy, and nonsmoking females with regular menstrual cycles were studied after they had given their informed consent and understood the experimental procedures. They did not engage in any regular physical activity for at least 1 year, and they recorded three consecutive menstrual cycles before entering this program. Ovulatory status was monitored by body temperature changes during the menstrual cycle. Plasma progesterone levels were measured to confirm the subjects’ actual menstrual phases during which exercise protocols were performed. They never took oral contraceptives and they abstained from any medication for at least 2 weeks before this study. Subjects were familiarized first with exercise on a bicycle ergometer (Corival 400) to eliminate the novel effect of a new experience. They then came to the laboratory four times (twice during midfollicular phases and twice during midluteal phases) to receive two different exercise protocols (strenuous exercise and moderate exercise).
Exercise and Blood Collection Protocol
All subjects arrived at 1 pm to participate in this study to avoid possible diurnal influences. After the subject had arrived at the laboratory and rested for 30 minutes, 35 mL of blood samples were drawn from a forearm vein for baseline data of hematological parameters and platelet function. Sodium citrate was used as an anticoagulant agent. Exercise began at 3:30 pm. The first exercise protocol consisted of 2 minutes of unloaded pedaling, followed by a continuous increment of workload of 10 to 20 W every 3 minutes until exhaustion (ie, strenuous exercise up to maximal oxygen consumption). Another exercise protocol was performed at about 50% of predetermined maximal oxygen consumption for 30 minutes (ie, moderate exercise). Immediately after exercise, another blood sample was collected for the measurements of postexercise hematological parameters and platelet function. Urine samples were collected before and 40 minutes after exercise. During exercise, the heart rate, minute ventilation, oxygen consumption, and CO2 production were obtained as described previously.6
Basic Hematological Parameters
Erythrocyte count, leukocyte count, platelet count, hematocrit, and hemoglobin concentration from the venous blood were determined by electronic counters (Cell Dyn 100 and 400, Metertech) as described in a previous study.6
A tapered parallel-plate chamber that provided shear-stress values covering the entire physiological range in human circulation was used to assess platelet adhesiveness on fibrinogen-coated glass, as described previously.6 13 A linear correlation between adherent platelets and local shear stress values was obtained. The slope of this line was used as an index of platelet adhesiveness (ie, the less negative the slope, the greater the platelet adhesiveness).
Platelet aggregation induced by ADP was evaluated by the percentage of reduction in single platelet counts as described in our previous study.6 Results were expressed as the percentage of aggregated platelets to total platelets, ie, (single platelet count before ADP−single platelet count after ADP)/(single platelet count before ADP)×100%.
Platelets were washed by repeated centrifugation with an albumin cushion and labeled with a calcium-sensitive fluorescent dye, fura-2 AM, as described before.14 [Ca2+]i levels were calculated from ratio values of fluorescence intensities measured at excitation wavelengths of 340 and 380 nm.15
Platelet Cyclic Nucleotides
Platelet cyclic nucleotides were determined by enzyme-linked immunosorbent assay using commercially available kits (Cayman, Ann Arbor, Mich).16 In brief, citrated platelet-rich plasma containing 10−4 mol/L of 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor, Sigma) was mixed with ice-cold HEPES buffer and centrifuged at 6 500×g at 4°C for 5 minutes. The pellet was vortexed with ice-cold 6% trichloroacetic acid for 2 minutes. After centrifugation (10 000×g, 15 minutes, 4°C), trichloroacetic acid was removed by washing four times with five volumes of water-saturated ether. The extracted samples were stored at −80°C until measurement.
Since PGI2 and thromboxane have short half-lives, we measured their stable urinary metabolites, ie, 6-keto-prostaglandin F1α and 11-dehydro-TXB2, respectively. These metabolites were measured by using commercial kits (Elisa Technologies, Lexington, Ky). The concentrations of these substances were normalized with urinary creatinine levels, which were determined by a modified Jaffe alkaline picrate method.17
Plasma NO Metabolites
NO metabolites in plasma were determined by the Griess reagent-based colorimetric method,18 using commercially available assay kits (Cayman, Ann Arbor, Mich). First, plasma nitrate was reduced to become nitrite. Then, nitrite was converted into a deep purple azo compound with an absorbance at 550 nm. Measured values represented the total amount of plasma NO metabolites, ie, nitrite plus nitrate.
Data were expressed as mean±SEM. The statistical software package of StatView IV on Macintosh was used for analysis of our data. The results were analyzed by two factorial analysis of variance followed by Tukey’s multiple comparison.19 Differences were considered significant at P<.05.
Anthropometric data of sedentary healthy females were as follows: age—22±1 year, height—161.1±1.5 cm, respectively. The body weight, resting heart rate, resting and blood pressure were not significantly different at various stages in females (53.5±1.2 kg, 73±1 bpm, 98/66±2 mm Hg for the midfollicular phase and 54.0±1.4 kg, 74±1 bpm, 98/63±1 mm Hg for the midluteal phase, respectively). Preexercise plasma progesterone levels at two different menstrual phases were as follows: 3.5±0.4 ng/mL before moderate exercise in the midfollicular phase, 3.1±0.3 ng/mL before severe exercise in the midfollicular phase, 31.6±3.7 ng/mL before moderate exercise in the midluteal phase, and 29.5±3.0 ng/mL before severe exercise in the midluteal phase, respectively. Neither their exercise performances nor their resting platelet functions were significantly different between these two menstrual phases (Tables 1⇓ and 2⇓).
Immediately after acute exercise either at the midfollicular phase or at the midluteal phase, the subjects showed increased levels of the hematological parameters in an intensity-dependent manner (Table 3⇓).
Platelet Adhesiveness and Aggregability
In the midfollicular phase, platelet adhesiveness was suppressed after moderate exercise and was elevated after strenuous exercise (Fig 1⇓). However, neither type of exercise had significant influence on platelet adhesiveness in the midluteal phase. Exercise-induced platelet adhesiveness changes were clearly different in these two menstrual phases.
Platelet aggregation induced by lower concentrations of ADP (0.25∼1 μmol/L) was enhanced after strenuous exercise and was decreased after moderate exercise in the midfollicular phase (Fig 2⇓). These exercise effects on platelet aggregability in the midfollicular phase were absent in the midluteal phase.
Basal platelet [Ca2+]i, 0.5 μmol/L, and 2 μmol/L ADP-evoked platelet [Ca2+]i were enhanced after strenuous exercise, and they were decreased after moderate exercise in the midfollicular phase (Fig 3⇓). These two types of exercise in the midluteal phase did not affect platelet [Ca2+]i.
Platelet Cyclic Nucleotides
Our results showed that platelet cGMP content drastically elevated after moderate exercise in the midfollicular phase but changed little after severe exercise (Fig 4⇓). In the midluteal phase, short-term exercise had no significant influence on platelet cGMP levels. Intracellular cAMP concentrations of platelets were not significantly influenced by exercise in either menstrual stage in females (Fig 4⇓).
Urinary Eicosanoids and Plasma NO Metabolites
Acute exercise apparently increased urinary 11-dehydro-TXB2 or 6-keto-prostaglandin F1α slightly (Fig 5⇓). In contrast, plasma NO metabolite levels more than doubled after moderate exercise in the midfollicular phase (Fig 5⇓). This is in accordance with the increase in platelet cGMP level under the same conditions (Fig 4⇑). Severe exercise in both phases or moderate exercise in midluteal phase had mild effects on plasma NO metabolite levels.
In this study, we have observed that (1) there were no significant differences in female exercise performance and resting platelet function between the midfollicular phase and the midluteal phase; (2) in the midluteal phase, short-term exercise did not affect platelet functions; (3) in the midfollicular phase, platelet functions were affected by short-term exercise in an intensity-dependent manner, ie, strenuous exercise potentiated platelets whereas moderate exercise suppressed them; (4) moderate exercise, but not severe exercise, during midfollicular phase significantly increased platelet cGMP content as well as plasma NO metabolite level; and (5) short-term exercise, irrespective of its intensity or during which phase of the menstrual cycle it was performed, minimally affected platelet cAMP content and urinary eicosanoid levels.
Perhaps the most surprising finding in this study is that female platelet-related parameters in response to exercise are totally different in two phases of the menstrual cycle. As a matter of fact, we have performed a similar study using female subjects without knowing at which point of their menstrual cycle the exercise was executed. No conclusion could be drawn from that study (unpublished data). Since short-term exercise did not affect platelet function during the midluteal phase, females in this phase should be less vulnerable to vigorous exercise-provoked thrombotic events. The incidence of cardiovascular diseases is more likely to be related to severe exercise than to either moderate exercise or resting state. Because women at the midluteal phase are not subjected to the adverse effects by strenuous exercise, they are better protected than men in this regard. As for the beneficial effects, they are most likely to be attained by long-term training with moderate exercise intensity. We have conducted such a study, and the results of men and women are similar20 (and unpublished data). Although the underlying mechanism responsible for these menstrual stage-dependent platelet responses to exercise remains to be investigated, our results may partially explain why premenopausal women have a lower incidence of cardiovascular diseases than men,5 at least during half of each month.
Epidemiological studies have suggested that adequate exercise may reduce the risk of cardiovascular disease whereas the risk of primary cardiac arrest may be transiently increased during vigorous exercise.3 4 21 This study is the first to report that the intensity of short-term exercise affects platelet function in females during midfollicular phase, ie, moderate exercise desensitizes platelets whereas strenuous exercise potentiates them. These findings on females during the midfollicular phase are similar to previous findings on male subjects.6 22 Taken together, our results could serve as one of several possible underlying mechanisms for explaining the epidemiological findings mentioned above.
Two previous studies on platelet function using female subjects at rest have reported controversial results regarding the effect of menstrual phases.23 24 Although both studies have shown insignificant changes in platelet count, volume, and aggregation, one study has found that plasma levels of β-thromboglobulin and platelet factor 4 (indicators for platelet release reaction) increase during ovulation and menstruation.23 However, this latter study did not calculate the ratio of these two proteins to rule out possible artifact due to blood sampling or handling.25 Our results are consistent with the idea that platelet functions are relatively constant in females at rest regardless of their menstrual phase.
NO is a potent antiplatelet agent that exerts its effects via the elevation of cGMP level.10 Our results showed that both plasma NO metabolites and platelet cGMP contents were increased by moderate exercise in the midfollicular phase. Moreover, under all other experimental conditions, platelet adhesiveness, aggregability, and [Ca2+]i were not suppressed. It is plausible to assume that moderate exercise induces NO release, probably from vascular endothelium, which elevates platelet cGMP and results in the desensitization of platelets. Acute exercise in rats has been reported to induce NO release and cause the attenuation of agonist-induced vasoconstrictive responses.9 However, severe exercise may potentiate platelets by dramatically elevating adrenaline, which is a known platelet-activating agent released during acute severe exercise,26 27 28 29 although plasma NO metabolites are also slightly increased after severe exercise (Fig 5⇑).
In conclusion, acute exercise affects female platelet function in an intensity-dependent manner in the midfollicular phase but not in the midluteal phase. These exercise-induced platelet functional changes may be mediated by NO.
Selected Abbreviations and Acronyms
|[Ca2+]i||=||intracellular calcium concentration|
This study was supported by the National Science Council in Taiwan, Republic of China (grants NSC84-2331-B-006-028 and NSC85-2331-B-006-046). We thank volunteers for their enthusiastic participation in this study.
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