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J Appl Physiol 98: 1414-1419, 2005; doi:10.1152/japplphysiol.00315.2004
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Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression

Antonino Coppola,1 Ludovico Coppola,1 Liliana dalla Mora,1 Francesco M. Limongelli,2 Antonio Grassia,1 Luigia Mastrolorenzo,1 Giorgio Gombos,2 and Giacomo Lucivero1

Departments of 1Geriatric Medicine and Metabolic Diseases and 2Experimental Medicine, Second University of Naples, Naples, Italy

Submitted 29 March 2004 ; accepted in final form 10 November 2004


   ABSTRACT
TOP
 ABSTRACT
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the "in vitro" ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.

sedentary men; active men; thromboregulation

IN EPIDEMIOLOGICAL studies, moderate and vigorous exercise have been associated with a decrease in the risk of cardiovascular disease (1, 16, 27, 41). However, vigorous exercise can acutely and transiently increase the risk of myocardial infarction and cardiac death, especially in sedentary subjects and in those with coronary heart disease (15, 31, 37, 43). The mechanism by which exercise triggers an acute cardiac event is not entirely known, although coronary thrombosis plays a key role. Several reports have demonstrated that strenuous exercise induces platelet activation, thus increasing thrombotic tendency (5, 22, 25, 33, 34). Various mechanisms, including increased plasma levels of norepinephrine and epinephrine (36), altered response of platelet {alpha}2-adrenergic receptors (39), and impaired sensitivity of platelets to nitric oxide (NO) (34), have been hypothesized to explain both the increased platelet activation and reactivity after strenuous exercise. The role of hemoconcentration induced by acute exercise also has been considered (8, 40). The epidemiology of coronary artery disease indicates that most myocardial infarctions occur in the early hours of the morning on arising (42). In the morning hours, patients are dehydrated; thereby circulating activated platelets are possibly more concentrated (32, 38).

Endothelial cell CD39/ATP diphosphohydrolase (ATPDase) (NTP diphosphohydrolase-1) has a critical role in inhibition of platelet responsiveness by regulating hydrolysis of extracellular adenine nucleotides (14, 28). CD39/ATPDase was originally identified as a lymphoid activation marker, and it is expressed on B lymphocytes, cytotoxic T lymphocytes, natural killer cells, monocytes, and endothelial cells (20, 23). The CD39/ATPDase on platelets and megakaryocytes was immunologically identified, and its ecto ATPDase activity was confirmed (23), although with levels much lower than in lymphocytes. Although no new data were published on megakaryiocytes, recently it was reported that platelet NTP diphosphohydrolase-1 activity is enhanced in Type 2 diabetic and hypertensive patients (26). More recently, the consequences of the transgenic expression of human CD39 in mice were described (9): human CD39 is widely expressed on the surface of mice platelets. These mice exhibited impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism.

Experimental data have shown the identity between CD39 and ADPase and, moreover, that this enzymatic activity is related to CD39 expression (28).

Considering the critical role of CD39 in thromboregulation mechanism, this study was designed to test the hypothesis that strenuous exercise could acutely modify CD39 expression on platelets and lymphocytes.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects.   Sixteen healthy men (eight sedentary and eight physically active subjects, matched for age and body mass index) were enrolled in this study. All subjects were volunteers and gave informed, written consent. The study was approved by the Ethics Committee of the School of Medicine of the Second University of Naples.

Basic characteristics of the examined subjects are shown in Table 1. The sedentary subjects did not engage in any regular physical activity for >1 yr before the study. The physically active subjects had engaged in regular physical activity at least three times a week. To prevent the confounding effect of smoking, all subjects were nonsmokers. Subjects with a history of ethanol use in excess of two drinks per day were excluded from the study. The volunteers were instructed to refrain from caffeine intake ≥12 h before and to take medications for at least 2 wk before the study.


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  		Table 1. Characteristics of the sedentary and physically active subjects participating in the study

 
Study protocol and preparation of blood samples.   All subjects were introduced to the bicycle ergometer exercise procedure on a separate day before baseline testing. This introductory session included getting practice on an electrically braked cycle and pedaling in the upright position.

On the experimental day, after the subject had arrived at the laboratory and rested for 30 min in the supine position, a blood sample was collected from an antecubital vein with no tourniquet to minimize platelet activation. The first 2 ml of blood were discarded, and 4.5 ml of blood were collected into a tube containing 0.5 ml of 3.8% sodium citrate. After 30 min of rest, the subject commenced exercise on a computerized bicycle ergometer (Cardioline ECT WS 2000, Stress Testing System, Remco Italia) with staring workload of 30 W and increments of 10 W/min. All subjects underwent electrocardiographic and blood pressure monitoring, and fatigue was estimated by the 20-grade Borg scale (2); exercise was terminated on exhaustion. Immediately after exercise, a second blood sample was collected, following the criteria previously described.

Blood samples, both at baseline and after exercise test, were examined within 10 min after drawing.

Flow cytometric analysis of platelets and leukocytes.   To evaluate the expression of glycoprotein (Gp) IIb-IIIA (PAC-1) and P-selectin (CD62P) on the platelet membrane, blood samples were prepared as described (12, 18). Within 10 min after drawing, 5 µl of citrated whole blood were added to 45 µl of HEPES-buffered saline containing 10 µl of anti-CD42a (clone Beb1) monoclonal antibody (MAb) conjugated to peridyn-chlorophyll protein (PerCP), 10 µl of anti-CD62P (clone AC1.2) MAb conjugated to phycoerythrin (PE), and 10 µl of anti-GpIIb/IIIa complex (clone PAC-1) MAb conjugated to FITC. Isotypic IgG1 MAb conjugated to PerCP and unrelated to human leukocyte antigens was used as negative control. All of the MAbs were purchased from Becton Dickinson (Milan, Italy). Blood samples were incubated for 20 min at room temperature without stirring and finally diluted gently with 450 µl of 1% cold paraformaldehyde in phosphate-buffered saline solution, supplemented with 0.2% sodium azide, to inhibit further platelet activation. Samples were stored at 4°C in the dark and analyzed within 24 h with a FACScan flow cytometer from Becton Dickinson.

To evaluate CD39 expression on platelets, whole blood samples were prepared as previously described and stained with 10 µl of PerCP-conjugated anti-CD42a MAb and 10 µl of anti-CD39 MAb (clone TU66) conjugated to PE (purchased from BD Pharmingen).

Whole blood flow cytometric techniques are used to minimize artifactual platelet activation, which can be caused in vitro by the centrifugation or filtration procedure used to separate platelets from other blood components (19, 22). To rule out that the platelet fraction was contaminated with monocytes and lymphocytes, which actually could have been responsible for the signal, we measured the CD39 in platelet-rich plasma (PRP) compared with platelet-poor plasma (PPP). PRP was prepared by centrifugation at 160 g for 10 min; platelets were washed twice by centrifugation at 1,400 g with 3.5 mM HEPES isosmolar buffer containing 142 mM NaCl, 2.5 mM KCl, and 5.5 mM glucose. The washed platelets were resuspended in HEPES isosmolar buffer, and platelet count was adjusted to values ranging from 300,000 to 400,000 platelets/mm3. The PPP was prepared by centrifugation at 1,400 g for 15 min.

The stained samples were analyzed at low flow rate with a FACScan cytometer after selection of logarithmic amplification for forward scatter (FSC) and side scatter (SSC) gains and by triggering acquisition of at least 10,000 CD42a (PerCP) positive events. By using the Cellquest software from Becton Dickinson, the multiparametric analysis of acquired events allows evaluation of the percentages of CD42a-positive platelets that coexpress P-selectin (CD62p), GpIIb/IIIA complex (PAC-1), or CD39 molecules and the mean fluorescence intensity of expression.

To determine, by flow cytometry (17), circulating platelet-platelet aggregates (PPAs), stained samples were further diluted with 1% paraformaldehyde in PBS before analysis at the flow cytometer to reduce the acquisition rate of CD42a (PerPC) positive platelets to <59 events/s and minimize the risk of platelet coincidence in the flow chamber. CD42a (PerCP) positive platelets were analyzed by FSC and SSC parameters; the percentages of CD42a-positive events exceeding the FSC and SSC values for single platelets were regarded as percentages of PPAs.

Expression of CD39 by T (CD3 positive) or B (CD19 positive) lymphocytes was evaluated by flow cytometry after staining whole blood samples with PerCP-conjugated anti-CD3, FITC-conjugated anti-CD19, and PE-conjugated anti-CD39. Anti-CD3 and anti-CD19 MAbs were purchased from Becton Dickinson. Briefly, 100 µl of whole blood were incubated with 10 µl of each MAb for 20 min at room temperature. Red blood cells were then lysed by 10-min incubation with 2 ml of FACS-lysing buffer (from Becton Dickinson), and the leukocyte pellet was recovered by centrifugation at 1,000 rpm for 5 min and two washings in 2 ml of PBS and was finally resuspended in 0.3 ml of PBS.

The percentages of T (CD3 positive) and B (CD19 positive) lymphocytes coexpressing CD39 were calculated by multiparametric analysis, as previously described.

ADP-induced platelet aggregation.   The "in vitro" platelet aggregation, induced by either 1.25 µM ADP and collagen (5 µg/ml), was measured according to Born (3), after adjusting platelet count in PRP to 350–400,000/µl. Data were recorded as maximum percentage of aggregation.

Red cell counts, total and differential leukocyte counts, platelet counts, and hematocrit were evaluated by using a JT2 model Coulter Counter (Instrumentation Laboratory, Milan, Italy).

Statistical analyses.   Data are presented as means with SD in parentheses. The nonparametric Mann-Whitney rank sum test was used to assess differences between the different substrates (i.e., whole blood, PRP, PPP) in the two groups (i.e., sedentary and physically active subjects) at baseline. ANOVA for repeated measures followed by two-tailed paired t-test was used to test for differences before and after vigorous exercise. Value of P < 0.05 was considered significant. The SigmaStat statistic program was used for all analyses.


   RESULTS
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Results of flow cytometric analysis of platelet CD39 expression in PRP, compared with both PPP and whole blood samples, are shown in Table 2. CD39 expression on both resting and activated platelets was significantly higher in PRP than in PPP and whole blood (P < 0.0001 and P < 0.001, respectively).


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  		Table 2. CD39 expression in resting and activated platelets

 
At baseline (Table 1), "in vitro" ADP- and collagen-induced platelet aggregation did not significantly differ between the two groups. PPAs were more numerous in the sedentary than in the active group [1.5 (SD 0.5) vs. 0.6% (SD 0.3); P < 0.05], whereas PAC-1 and CD39 were higher in sedentary than in the active group [PAC-1: 4.6 (SD 3.5) vs. 2.3% (SD 2.1), P < 0.05; CD39: 2.2 (SD 0.8) vs. 0.6% (SD 0.2), P < 0.01]; the addition of ADP to samples resulted in significant increase of CD39 expression in platelet surface in both groups [sedentary subjects: from 2.2 (SD 0.8) to 3.4% (SD 1.2), P < 0.001; active subjects: from 0.6 (SD 0.2) to 1.5% (SD 0.5), P < 0.001]. The expression of platelet CD62P in nonstimulated samples did not statistically differ between active and sedentary subjects. The same is true for the stimulated samples. Also, the expression of CD39 in B and in T lymphocytes did not statistically differ between active and sedentary subjects.

Immediately after strenuous exercise, both sedentary and active subjects demonstrated statistically significant platelet activation as judged by the percentage of circulating PPAs [sedentary subjects: from 1.5 (SD 0.5) to 2.1% (SD 0.6), P < 0.01; active subjects: from 0.6 (SD 0.3) to 0.8% (SD 0.2), P < 0.05] (Fig. 1). The in vitro ADP-induced platelet aggregation significantly increased in sedentary subjects but not physically active subjects [sedentary subjects: from 27 (SD 13) to 36% (SD 9), P < 0.01; active subjects: from 25 (SD 9.4) to 27% (SD 11), not significant] (Fig. 2). Exercise also significantly increased the expression of CD62P on ADP-stimulated platelet surface but only in sedentary subjects [from 46 (SD 10) to 55% (SD 8), P < 0.05] (Table 3).



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  		Fig. 1. Platelet-platelet aggregates (PPAs) after vigorous exercise. Values are means ± SD. P values are shown.

 


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  		Fig. 2. Representative platelet-aggregation tracings obtained on platelet-rich plasma from a sedentary (A) and a physically active (B) subject before and after vigorous exercise.

 

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  		Table 3. Effects of vigorous exercise on platelet activation state and CD39 expression in sedentary and physically active subjects

 
After exercise, there was a significant reduction of CD39 expression in platelets [sedentary subjects: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P < 0.01; active subjects: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P < 0.01] (Fig. 3) and an increase of CD39 expression in B lymphocytes [sedentary subjects: from 47 (SD 13) to 60% (SD 11), P < 0.005; active subjects: from 46 (SD 11) to 59% (SD 11), P < 0.005] (Fig. 4).



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  		Fig. 3. Effects of vigorous exercise on platelet CD39 expression. Values are means ± SD. P values are shown.

 


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  		Fig. 4. Effect of vigorous exercise on B lymphocytes CD39 expression. Values are means ± SD.

 
In both groups (sedentary and active subjects), expression of PAC-1 on platelets and CD39 expression in T lymphocytes did not change significantly following strenuous exercise (Table 3).


   DISCUSSION
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first work evaluating the acute effects of strenuous exercise on CD39 expression in platelets and lymphocytes.

The results of flow cytometric analysis of platelet CD39 expression in PRP, compared with PPP, demonstrate that CD39 is actually present in platelets. In addition, the level of CD39 expression in PRP, higher than that in whole blood sample, suggests that the repeated centrifugations activated the platelets (22, 29).

In agreement with previous reports, our results seem to confirm that strenuous exercise in sedentary subjects and, to a lesser extent, in physically active subjects induces platelet activation (22, 25, 33, 34, 39).

The novel result of the present study is that strenuous exercise reduces CD39 expression in platelets and increases it in B lymphocytes. Taken together, these findings confirm the critical role of this ADPase in regulating platelet responsiveness and suggest a possible role of B lymphocytes in thromboregulation mechanism.

On stimulation, platelets release ATP, ADP, serotonin, thromboxane A2, and other biologically active compounds, which, in turn, further activate platelets, endothelial cells, and neutrophils into the evolving thrombus (29). Nucleotides are the most important platelet agonists, inducing activation, recruitment, and aggregation of platelets (7, 13). The CD39/ATPDase in endothelial cells has a critical role in the inhibition of platelet responsiveness by keeping low the levels of adenine nucleotides (28). The local generation and the release of PGI2 and NO are the other mechanisms by which endothelial cells modulate blood fluidity (4, 30). A possible role of platelet and lymphoid cells CD39/ADPase in inhibition of platelet responsiveness has not yet been considered.

Several epidemiological studies have demonstrated that physical activity reduces the risk of cardiovascular disease (1, 16, 27, 41). However, exercise presents risks as well as benefits. In fact, exercise has long been thought a precipitating factor for cardiac events (31, 37, 43). The risks of complications are highest in habitually inactive individuals during unaccustomed vigorous exercise, especially in those with coronary heart disease or with more cardiac risk factors (15). Previous data have shown that strenuous exercise increases the thrombotic tendency, thereby providing a mechanism by which exercise could trigger cardiac events (22, 25, 33, 34).

In the present study, the platelet response to strenuous exercise to cycle ergometry test has been investigated by evaluating platelet aggregation, the PPAs, activation of platelet {alpha}IIb-{beta}3 receptor, and exposure of P-selectin. Both of these glycoproteins are considered markers of activated platelets and are involved in aggregation and adhesive spreading during hemostasis. In agreement with other authors (22, 25, 33, 34, 39), we found that exercise induces a platelet reactivity and activation state, as reflected by enhanced platelet sensitivity to in vitro stimulation, elevated circulating PPA counts, and increased exposure of P-selectin recognized by flow cytometry using CD62P antibodies. These responses were more evident in sedentary than in physically active subjects. Various mechanisms, including impaired sensitivity to NO and altered performance of platelet {alpha}2-adrenergic receptors, have been hypothesized to explain the changes of platelet function induced by strenuous exercise, especially in sedentary subjects (34, 39).

A novel aspect of our data is the demonstration that CD39 expression decreased in platelets and increased in B lymphocytes after strenuous exercise. The role of B lymphocytes in the pathogenesis of vascular thrombosis and atherosclerosis remains unclear, although recent reports provide evidence for a protective role through different mechanisms (6, 17).

We hypothesize that a decreased CD39/ATPDase expression by platelets indicates its consumption or shedding in thromboregulation. Shedding of CD39/ATPDase, which may remain enzymatically active, was already previously reported in thrombin-treated cells (35).

Most likely, on analogy of what was suggested by other authors for the increase both of prostacyclin biosynthesis (33) and of platelet NO release (21), our finding that B-lymphocyte CD39 expression increased may represent a compensatory response and may play a protective role against exercise-induced activation of platelets.

The increase of CD39 expression on platelet surface that we found after the in vitro addition of ADP in both sedentary and active subject groups is, for the moment, difficult to interpret. Because platelets are anucleate, the possibility of upregulation of the enzyme synthesis is excluded (18). The increased expression of CD39 could be due to increased exposure of "masked" molecules and/or to translocation/activation of an inactive intracellular precursor molecule provoked by the ADP binding to the plasma membrane (44). These phenomena may be associated with the platelet shape change that immediately follows the binding of the agonist to platelet surface receptors (3).

On the other hand, data from recent studies in rats show that different agents (free radicals, antioxidant) (10, 12) and different experimental conditions (ischemia, reperfusion, and ischemic preconditioning) (11) alter platelet ATPDase activity.

In conclusion, our results support the hypothesis that strenuous physical exercise acutely modifies the expression of platelet and B-lymphocyte CD39/ATPDase. Further studies are necessary to evaluate whether these modifications, possibly by altering the platelet-platelet and platelet-endothelial cell resting balance, concur to the exercise-related enhanced risk of acute cardiac events.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Coppola, Divisione di Astanteria Medica e Geriatria, Policlinico Universitario, Piazza Miraglia 2, 80138 Napoli, Italy (E-mail: ludovico.coppola{at}unina2.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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